Rajalakshmi Engineering College

Department of Biotechnology
Faculty Name : Mr.M.Sankar !ecturer"
Staff co#e: B$%% Semester :&'' SEC ()B
IMMUNOTECHNOLOGY BT2046
UNIT I ANTIGENS
(n antigen is a su*stance+molecule that ,hen intro#uce# into the *o#y triggers the pro#uction
of an anti*o#y *y the immune system ,hich ,ill then kill or neutrali-e the antigen that is
recogni-e# as a foreign an# potentially harmful in.a#er. $hese in.a#ers can *e molecules such
as pollen or cells such as *acteria. /riginally the term came from anti*o#y generator an# ,as a
molecule that *in#s specifically to an anti*o#y0 *ut the term no, also refers to any molecule or
molecular fragment that can *e *oun# *y a major histocompati*ility comple1 M2C" an#
presente# to a $3cell receptor4Self4 antigens are usually tolerate# *y the immune system5
,hereas 4Non3self4 antigens are i#entifie# as intru#ers an# attacke# *y the immune system.
(utoimmune #isor#ers arise from the immune system reacting to its o,n antigens.
Antigen
Each anti*o#y *in#s to a specific antigen5 an interaction similar to a lock an# key.
Similarly0 an immunogen is a specific type of antigen. (n immunogen is #efine# as a su*stance
that is a*le to pro.oke an a#apti.e immune response if injecte# on its o,n.Sai# another ,ay0 an
immunogen is a*le to in#uce an immune response0 ,hile an antigen is a*le to com*ine ,ith the
pro#ucts of an immune response once they are ma#e. $he o.erlapping concepts of
immunogenicity an# antigenicity are there*y su*tly #ifferent. (ccor#ing to a current te1t *ook:
'mmunogenicity is the a*ility to in#uce a humoral an#+or cell3me#iate# immune response
(ntigenicity is the a*ility to com*ine specifically ,ith the final pro#ucts of the 6immune
response7 i.e. secrete# anti*o#ies an#+or surface receptors on $3cells". (lthough all molecules
that ha.e the property of immunogenicity also ha.e the property of antigenicity0 the re.erse is
not true.4
(t the molecular le.el0 an antigen is characteri-e# *y its a*ility to *e 4*oun#4 at the antigen3
*in#ing site of an anti*o#y. Note also that anti*o#ies ten# to #iscriminate *et,een the specific
molecular structures presente# on the surface of the antigen as illustrate# in the Figure".
(ntigens are usually proteins or polysacchari#es. $his inclu#es parts coats0 capsules0 cell ,alls0
flagella0 fim*rae0 an# to1ins" of *acteria0 .iruses0 an# other microorganisms. !ipi#s an# nucleic
aci#s are antigenic only ,hen com*ine# ,ith proteins an# polysacchari#es. Non3micro*ial
e1ogenous non3self" antigens can inclu#e pollen0 egg ,hite0 an# proteins from transplante#
tissues an# organs or on the surface of transfuse# *loo# cells. &accines are e1amples of
immunogenic antigens intentionally a#ministere# to in#uce ac8uire# immunity in the recipient.
Cells present their immunogenic3antigens to the immune system .ia a histocompati*ility
molecule. Depen#ing on the antigen presente# an# the type of the histocompati*ility molecule0
se.eral types of immune cells can *ecome acti.ate#.
Related concepts
Eitoe 3 $he #istinct molecular surface features of an antigen capa*le of *eing *oun#
*y an anti*o#y a.k.a. antigenic determinant". (ntigenic molecules0 normally *eing
4large4 *iological polymers0 usually present se.eral surface features that can act as points
of interaction for specific anti*o#ies. (ny such #istinct molecular feature constitutes an
epitope. Most antigens therefore ha.e the potential to *e *oun# *y se.eral #istinct
anti*o#ies0 each of ,hich is specific to a particular epitope. 9sing the 4lock an# key4
metaphor0 the antigen itself can *e seen as a string of keys 3 any epitope *eing a 4key4 3
each of ,hich can match a #ifferent lock. Different anti*o#y i!iotye"0 each ha.ing
#istinctly forme# complementarity #etermining regions0 correspon# to the .arious
4locks4 that can match 4the keys4 epitopes" presente# on the antigen molecule.
A##e$gen 3 ( su*stance capa*le of causing an allergic reaction. $he #etrimental"
reaction may result after e1posure .ia ingestion0 inhalation0 injection0 or contact ,ith
skin.
Sue$antigen 3 ( class of antigens ,hich cause non3specific acti.ation of $3cells
resulting in polyclonal $ cell acti.ation an# massi.e cytokine release.
To#e$ogen 3 ( su*stance that in.okes a specific immune non3responsi.eness #ue to its
molecular form. 'f its molecular form is change#0 a tolerogen can *ecome an
immunogen.
Immunog#o%u#in %in!ing $otein 3 $hese proteins are capa*le of *in#ing to anti*o#ies
at positions outsi#e of the antigen3*in#ing site. $hat is0 ,hereas antigens are the 4target4
of anti*o#ies0 immunoglo*ulin *in#ing proteins 4attack4 anti*o#ies. :rotein (0 protein ;
an# protein ! are e1amples of proteins that strongly *in# to .arious anti*o#y isotypes.
Origin of the term antigen
'n <=>> !a#islas Deutsch !as-lo Detre" <=?@A<>%>" name# the hypothetical su*stances
half,ay *et,een *acterial constituents an# anti*o#ies 4su*stances immunogenes ou antigenes4.
2e originally *elie.e# those su*stances to *e precursors of anti*o#ies0 just like -ymogen is a
precursor of -ymase. But *y <>B% he un#erstoo# that an antigen in#uces the pro#uction of
immune *o#ies anti*o#ies" an# ,rote that the ,or# antigen ,as a contraction of
4(ntisomatogen C 'mmunkDrper*il#ner4. $he /1for# English Dictionary in#icates that the
logical construction shoul# *e 4anti*o#y"3gen4
6E7
.
Classification of antigens
(ntigens can *e classifie# in or#er of their class.
E&ogenou" antigen"
E1ogenous antigens are antigens that ha.e entere# the *o#y from the outsi#e0 for e1ample *y
inhalation0 ingestion0 or injection. $he immune systemFs response to e1ogenous antigens is often
su*clinical. By en#ocytosis or phagocytosis0 e1ogenous antigens are taken into the antigen3
presenting cells (:Cs" an# processe# into fragments. (:Cs then present the fragments to $
helper cells CD@
G
" *y the use of class '' histocompati*ility molecules on their surface. Some $
cells are specific for the pepti#e:M2C comple1. $hey *ecome acti.ate# an# start to secrete
cytokines. Cytokines are su*stances that can acti.ate cytoto1ic $ lymphocytes C$!"0 anti*o#y3
secreting B cells0 macrophages0 an# other particles.
Some antigens start out as e1ogenous antigens0 an# later *ecome en#ogenous for e1ample0
intracellular .iruses". 'ntracellular antigens can *e release# *ack into circulation upon the
#estruction of the infecte# cell0 again.
En!ogenou" antigen"
En#ogenous antigens are antigens that ha.e *een generate# ,ithin pre.iously normal cells as a
result of normal cell meta*olism0 or *ecause of .iral or intracellular *acterial infection. $he
fragments are then presente# on the cell surface in the comple1 ,ith M2C class ' molecules. 'f
acti.ate# cytoto1ic CD=
G
$ cells recogni-e them0 the $ cells *egin to secrete .arious to1ins that
cause the lysis or apoptosis of the infecte# cell. 'n or#er to keep the cytoto1ic cells from killing
cells just for presenting self3proteins0 self3reacti.e $ cells are #elete# from the repertoire as a
result of tolerance also kno,n as negati.e selection". En#ogenous antigens inclu#e 1enogenic
heterologous"0 autologous an# i#iotypic or allogenic homologous" antigens.
Autoantigen"
(n autoantigen is usually a normal protein or comple1 of proteins an# sometimes DN( or
RN(" that is recogni-e# *y the immune system of patients suffering from a specific autoimmune
#isease. $hese antigens shoul#0 un#er normal con#itions0 not *e the target of the immune
system0 *ut0 #ue to mainly genetic an# en.ironmental factors0 the normal immunological
tolerance for such an antigen has *een lost in these patients.
Tumor antigens
Tumor antigens or neoantigens are
6citation needed7
those antigens that are presente# *y M2C ' or
M2C '' molecules on the surface of tumor cells. $hese antigens can sometimes *e presente# *y
tumor cells an# ne.er *y the normal ones. 'n this case0 they are calle# tumor3specific antigens
$S(s" an#0 in general0 result from a tumor3specific mutation. More common are antigens that
are presente# *y tumor cells an# normal cells0 an# they are calle# tumor3associate# antigens
$((s". Cytoto1ic $ lymphocytes that recogni-e these antigens may *e a*le to #estroy the
tumor cells *efore they proliferate or metastasi-e.
$umor antigens can also *e on the surface of the tumor in the form of0 for e1ample0 a mutate#
receptor0 in ,hich case they ,ill *e recogni-e# *y B cells.
Nativity
( nati'e antigen is an antigen that is not yet processe# *y an (:C to smaller parts. $ cells
cannot *in# nati.e antigens0 *ut re8uire that they *e processe# *y (:Cs0 ,hereas B cells can *e
acti.ate# *y nati.e ones.
Antigenic specificity
Antigen(ic) "eci*icity is the a*ility of the host cells to recogni-e an antigen specifically as a
uni8ue molecular entity an# #istinguish it from another ,ith e18uisite precision. (ntigen
specificity is #ue primarily to the si#e3chain conformations of the antigen. 't is a measurement0
although the #egree of specificity may not *e easy to measure0 an# nee# not *e linear or of the
nature of a rate3limite# step or e8uation.
St$uctu$e o* antigen
+$ea$ation O* Antigen" ,o$ -ai"ing Anti%o!ie"
+o#yc#ona# anti%o!ie" or antisera" are anti*o#ies that are o*taine# from #ifferent B cell
resources. $hey are a com*ination of immunoglo*ulin molecules secrete# against a specific
antigen0 each i#entifying a #ifferent epitope.
Production
$hese anti*o#ies are typically pro#uce# *y immuni-ation of a suita*le mammal0 such as a
mouse0 ra**it or goat. !arger mammals are often preferre# as the amount of serum that can *e
collecte# is greater. (n antigen is injecte# into the mammal. $his in#uces the B3lymphocytes to
pro#uce 'g; immunoglo*ulins specific for the antigen. $his polyclonal 'g; is purifie# from the
mammalHs serum.By contrast0 monoclonal anti*o#ies are #eri.e# from a single cell line.
Many metho#ologies e1ist for polyclonal anti*o#y pro#uction in la*oratory animals.
'nstitutional gui#elines go.erning animal use an# proce#ures relating to these metho#ologies are
generally oriente# aroun# humane consi#erations an# appropriate con#uct for a#ju.ant agents
,hich mo#ify the effect of other agents ,hile ha.ing fe, if any #irect effects ,hen gi.en *y
themsel.es" use. $his inclu#es a#ju.ant selection0 routes an# sites of a#ministration0 injection
.olumes per site an# num*er of sites per animal. 'nstitutional policies generally inclu#e
allo,a*le .olumes of *loo# per collection an# safety precautions inclu#ing appropriate restraint
an# se#ation or anesthesia of animals for injury pre.ention to animals or personnel.
$he primary goal of anti*o#y pro#uction in la*oratory animals is to o*tain high titer0 high
affinity antisera for use in e1perimentation or #iagnostic tests. (#ju.ants are use# to impro.e or
enhance an immune response to antigens. Most a#ju.ants pro.i#e for an injection site0 antigen
#epot ,hich allo,s for a slo, release of antigen into #raining lymph no#es.
Many a#ju.ants also contain or act #irectly as:
<. surfactants ,hich promote concentration of protein antigens molecules o.er a large
surface area0 an#
I. immunostimulatory molecules or properties. (#ju.ants are generally use# ,ith solu*le
protein antigens to increase anti*o#y titers an# in#uce a prolonge# response ,ith
accompanying memory.
Such antigens *y themsel.es are generally poor immunogens. Most comple1 protein antigens
in#uce multiple B3cell clones #uring the immune response0 thus0 the response is polyclonal.
'mmune responses to non3protein antigens are generally poorly or enhance# *y a#ju.ants an#
there is no system memory.
(nti*o#ies are currently also *eing pro#uce# from isolation of human B3lymphocytes to
pro#uce specific recom*inant polyclonal anti*o#ies. $he *iotechnology company0 Symphogen0
pro#uces this type of anti*o#y for therapeutic applications. $hey are the first research company
to #e.elop recom*inant polyclonal anti*o#y #rugs to reach phase t,o trials. $his pro#uction
pre.ents .iral an# prion transmission.
Animal selection
(nimals fre8uently use# for polyclonal anti*o#y pro#uction inclu#e chickens0 goats0 guinea
pigs0 hamsters0 horses0 mice0 rats0 an# sheep. 2o,e.er0 the ra**it is the most commonly use#
la*oratory animal for this purpose. (nimal selection shoul# *e *ase# upon:
<. the amount of anti*o#y nee#e#0
I. the relationship *et,een the #onor of the antigen an# the recipient anti*o#y pro#ucer
generally the more #istant the phylogenetic relationship0 the greater the potential for
high titer anti*o#y response" an#
%. the necessary characteristics 6e.g.0 class0 su*class isotype"0 complement fi1ing nature7 of
the anti*o#ies to *e ma#e. 'mmuni-ation an# phle*otomies are stress associate# an#0 at
least ,hen using ra**its an# ro#ents0 specific pathogen free S:F" animals are preferre#.
9se of such animals can #ramatically re#uce mor*i#ity an# mortality #ue to pathogenic
organisms0 especially Pasteurella multocida in ra**its.
;oats or horses are generally use# ,hen large 8uantities of antisera are re8uire#. Many
in.estigators fa.or chickens *ecause of their phylogenetic #istance from mammals. Chickens
transfer high 8uantities of 'gJ 'g;" into the egg yolk an# har.esting anti*o#ies from eggs
eliminates the nee# for the in.asi.e *lee#ing proce#ure. /ne ,eekHs eggs can contain <B times
more anti*o#ies than the .olume of ra**it *loo# o*taine# from one ,eekly *lee#ing. 2o,e.er0
there are some #isa#.antages ,hen using certain chicken #eri.e# anti*o#ies in immunoassays.
Chicken 'gJ #oes not fi1 mammalian complement component C< an# it #oes not perform as a
precipitating anti*o#y using stan#ar# solutions.
(lthough mice are use# most fre8uently for monoclonal anti*o#y pro#uction0 their small si-e
usually pre.ents their use for sufficient 8uantities of polyclonal0 serum anti*o#ies. 2o,e.er0
polyclonal anti*o#ies in mice can *e collecte# from ascites flui# using any one of a num*er of
ascites pro#ucing metho#ologies.
Khen using ra**its0 young a#ult animals I.LA%.B kg or L.L3E.Ll*s" shoul# *e use# for primary
immuni-ation *ecause of the .igorous anti*o#y response. 'mmune function peaks at pu*erty an#
primary responses to ne, antigens #ecline ,ith age. Female ra**its are generally preferre#
*ecause they are more #ocile an# are reporte# to mount a more .igorous immune response than
males. (t least t,o animals per antigen shoul# *e use# ,hen using out*re# animals. $his
principle re#uces potential total failure resulting from non3responsi.eness to antigens of
in#i.i#ual animals.
Antigen preparation
$he si-e0 e1tent of aggregation an# relati.e nati.ity of protein antigens can all #ramatically
affect the 8uality an# 8uantity of anti*o#y pro#uce#. Small polypepti#es M<B ku" an# non3
protein antigens generally nee# to *e conjugate# or crosslinke# to larger0 immunogenic0 carrier
proteins to increase immunogenicity an# pro.i#e $ cell epitopes. ;enerally0 the larger the
immunogenic protein the *etter. !arger proteins0 e.en in smaller amounts0 usually result in *etter
engagement of antigen presenting antigen processing cells for a satisfactory immune response.
'njection of solu*le0 non3aggregate# proteins has a higher pro*a*ility of in#ucing tolerance
rather than a satisfactory anti*o#y response.
Neyhole limpet hemocyanin N!2" an# *o.ine serum al*umin are t,o ,i#ely use# carrier
proteins. :oly3!3lysine has also *een use# successfully as a *ack*one for pepti#es. (lthough the
use of :oly3!3lysine re#uces or eliminates pro#uction of anti*o#ies to foreign proteins0 it may
result in failure of pepti#e3in#uce# anti*o#y pro#uction. Recently0 liposomes ha.e also *een
successfully use# for #eli.ery of small pepti#es an# this techni8ue is an alternati.e to #eli.ery
,ith oily emulsion a#ju.ants.
Antigen .uantity
Selection of antigen 8uantity for immuni-ation .aries ,ith the properties of the antigen an# the
a#ju.ant selecte#. 'n general0 microgram to milligram 8uantities of protein in a#ju.ant are
necessary to elicit high titer anti*o#ies. (ntigen #osage is generally species0 rather than *o#y
,eight0 associate#. $he so calle# O,in#o,P of immunogenicity in each species is *roa# *ut too
much or too little antigen can in#uce tolerance0 suppression or immune #e.iation to,ar#s
cellular immunity rather than a satisfactory humoral response. /ptimal an# usual protein antigen
le.els for immuni-ing specific species ha.e *een reporte# in the follo,ing ranges:
<. ra**it0 LBA<BBB Qg5
I. mouse0 <BAIBB Qg5
%. guinea pig0 LBALBB Qg5 an#
@. goat0 ILBALBBB Qg.
/ptimal OprimingP #oses are reporte# to *e at the lo, en# of each range.
$he affinity of serum anti*o#ies increases ,ith time months" after injection of antigen3a#ju.ant
mi1tures an# as antigen in the system #ecreases. Ki#ely use# antigen #osages for O*oosterP or
secon#ary immuni-ations are usually one half to e8ual the priming #osages. (ntigens shoul# *e
free of preparati.e *ypro#ucts an# chemicals such as polyacrylami#e gel0 SDS0 urea0 en#oto1in0
particulate matter an# e1tremes of p2.
+eti!e Anti%o!ie"
Khen a pepti#e is *eing use# to generate the anti*o#y0 it is e1tremely important to #esign the
antigens properly. $here are se.eral resources that can ai# in the #esign as ,ell as companies
that offer this ser.ice. E1pasy has aggregate# a set of pu*lic tools un#er its :rotScale page that
re8uire some #egree of user kno,le#ge to na.igate. For a more simple pepti#e scoring tool there
is a (ntigen :rofiler tool a.aila*le that ,ill ena*le you to score in#i.i#ual pepti#e se8uences
*ase# upon a relation epitope mapping #ata*ase of pre.ious immunogens use# to generate
anti*o#ies. Finally0 as a general rule pepti#es shoul# follo, some *asic criteria.
Khen e1amining pepti#es for synthesis an# immuni-ation0 it is recommen#e# that certain
resi#ues an# se8uences *e a.oi#e# #ue to potential synthesis pro*lems. $his inclu#es some of
the more common characteristics:
E1tremely long repeats of the same amino aci# e.g. RRRR"
Serine S"0 $hreonine $"0 (lanine ("0 an# &aline &" #ou*lets
En#ing or starting a se8uence ,ith a proline :"
;lutamine R" or (sparagine N" at the n3terminus
:epti#es o.er ,eighte# ,ith hy#ropho*ic resi#ues e.g. &0(0!0'0 etcS"
-eacti'ity
'n.estigators shoul# also consi#er the status of nati.ity of protein antigens ,hen use# as
immunogens an# reaction ,ith anti*o#ies pro#uce#. (nti*o#ies to nati.e proteins react *est ,ith
nati.e proteins an# anti*o#ies to #enature# proteins react *est ,ith #enature# proteins. 'f elicite#
anti*o#ies are to *e use# on mem*rane *lots proteins su*jecte# to #enaturing con#itions" then
anti*o#ies shoul# *e ma#e against #enature# proteins. /n the other han#0 if anti*o#ies are to *e
use# to react ,ith a nati.e protein or *lock a protein acti.e site0 then anti*o#ies shoul# *e ma#e
against the nati.e protein. (#ju.ants can often alter the nati.ity of the protein. ;enerally0
a*sor*e# protein antigens in a preforme# oil3in3,ater emulsion a#ju.ant0 retain greater nati.e
protein structure than those in ,ater3in3oil emulsions.
A"eticity
(ntigens shoul# al,ays *e prepare# using techni8ues that ensure that they are free of micro*ial
contamination. Most protein antigen preparations can *e sterili-e# *y passage through a B.IIu
filter. Septic a*scesses often occur at inoculation sites of animals ,hen contaminate#
preparations are use#. $his can result in failure of immuni-ation against the targete# antigen.
Adjuvants
$here are many commercially a.aila*le immunologic a#ju.ants. Selection of specific a#ju.ants
or types .aries #epen#ing upon ,hether they are to *e use# for research an# anti*o#y pro#uction
or in .accine #e.elopment. (#ju.ants for .accine use only nee# to pro#uce protecti.e anti*o#ies
an# goo# systemic memory ,hile those for antiserum pro#uction nee# to rapi#ly in#uce high
titer0 high a.i#ity anti*o#ies. No single a#ju.ant is i#eal for all purposes an# all ha.e a#.antages
an# #isa#.antages. (#ju.ant use generally is accompanie# *y un#esira*le si#e effects of .arying
se.erity an# #uration. Research on ne, a#ju.ants focuses on su*stances ,hich ha.e minimal
to1icity ,hile retaining ma1imum immunostimulation. 'n.estigators shoul# al,ays *e a,are of
potential pain an# #istress associate# ,ith a#ju.ant use in la*oratory animals.
$he most fre8uently use# a#ju.ants for anti*o#y pro#uction are Freun#Hs0 (lum0 the Ri*i
(#ju.ant System an# $iterma1.
,$eun!/" a!0u'ant"
$here are t,o *asic types of Freun#Fs a#ju.ants: Freun#Hs Complete (#ju.ant FC(" an#
Freun#Hs 'ncomplete (#ju.ant F'(". FC( is a ,ater3in3oil emulsion that locali-es antigen for
release perio#s up to E months. 't is formulate# ,ith mineral oil0 the surfactant manni#e
monoleate an# heat kille# Mycobacterium tuberculosis0 Mycobacterium butyricum or their
e1tracts for aggregation of macrophages at the inoculation site". $his potent a#ju.ant stimulates
*oth cell me#iate# an# humoral immunity ,ith preferential in#uction of anti*o#y against
epitopes of #enature# proteins. (lthough FC( has historically *een the most ,i#ely use#
a#ju.ant0 it is one of the more to1ic agents #ue to non3meta*oli-a*le mineral oil an# it in#uces
granulomatous reactions. 'ts use is limite# to la*oratory animals an# it shoul# *e use# only ,ith
,eak antigens. 't shoul# not *e use# more than once in a single animal since multiple FC(
inoculations can cause se.ere systemic reactions an# #ecrease# immune responses. Freun#Hs
'ncomplete (#ju.ant has the same formulation as FC( *ut #oes not contain myco*acterium or
its components. F'( usually is limite# to *ooster #oses of antigen since it normally much less
effecti.e than FC( for primary anti*o#y in#uction. Freun#Hs a#ju.ants are normally mi1e# ,ith
e8ual parts of antigen preparations to form sta*le emulsions.
-i%i A!0u'ant Sy"tem
Ri*i a#ju.ants are oil3in3,ater emulsions ,here antigens are mi1e# ,ith small .olumes of a
meta*oli-a*le oil s8ualene" ,hich are then emulsifie# ,ith saline containing the surfactant
$,een =B. $his system also contains refine# myco*acterial pro#ucts cor# factor0 cell ,all
skeleton" as immunostimulants an# *acterial monophosphoryl lipi# (. $hree #ifferent species
oriente# formulations of the a#ju.ant system are a.aila*le. $hese a#ju.ants interact ,ith
mem*ranes of immune cells resulting in cytokine in#uction0 ,hich enhances antigen uptake0
processing an# presentation. $his a#ju.ant system is much less to1ic an# less potent than FC(
*ut generally in#uces satisfactory amounts of high a.i#ity anti*o#ies against protein antigens.
Tite$ma&
$iterma1 represents a ne,er generation of a#ju.ants that are less to1ic an# contain no
*iologically #eri.e# materials. 't is *ase# upon mi1tures of surfactant acting0 linear0 *locks or
chains of nonionic copolymers polyo1ypropylene :/:" an# polyo1yethylene :/E". $hese
copolymers are less to1ic than many other surfactant materials an# ha.e potent a#ju.ant
properties ,hich fa.or chemota1is0 complement acti.ation an# anti*o#y pro#uction. $iterma1
a#ju.ant forms a microparticulate ,ater3in3oil emulsion ,ith a copolymer an# meta*oli-a*le
s8ualene oil. $he copolymer is coate# ,ith emulsion sta*ili-ing silica particles ,hich allo,s for
incorporation of large amounts of a ,i#e .ariety of antigenic materials. $he a#ju.ant acti.e
copolymer forms hy#rophilic surfaces0 ,hich acti.ate complement0 immune cells an# increase#
e1pression of class '' major histocompati*ility molecules on macrophages. $iterma1 presents
antigen in a highly concentrate# form to the immune system0 ,hich often results in anti*o#y
titers compara*le to or higher than FC(.
Seco#: Specol is a ,ater in oil a#ju.ant ma#e of purifie# mineral oil. 't has *een reporte# to
in#uce immune response compara*le to Freun#Fs a#ju.ant in ra**it an# other research animal
,hile pro#ucing fe,er histological lesions
A!0u'ant" an! t1ei$ mo!e" o* action
$he tren# to,ar#s the use of pepti#es an# su*unit proteins in mo#ern .accine #esign has
necessitate# the use of immunological a#ju.ants to achie.e effecti.e immunity. (luminium
hy#ro1i#e0 a component of the #iphtheria0 tetanus an# hepatitis B .accines0 ,as first #escri*e#
as an a#ju.ant o.er EB years ago an# is the only a#ju.ant currently appro.e# for use in humans.
't is also a common component of many .eterinary .accines. Khile this a#ju.ant is effecti.e at
enhancing anti*o#y titres to antigens0 the effecti.eness of aluminium hy#ro1i#e is limite# #ue to
its ina*ility to promote cell me#iate# immunity. Freun#Fs Complete (#ju.ant FC(" has *een
use# e1perimentally an# #oes stimulate cellular immunity0 *ut is unsuita*le for human an#
.eterinary use as it promotes0 amongst other to1ic si#e effects0 local inflammation an#
granuloma formation at the site of injection. $hus0 in recent years there has *een a great #eal of
interest in #e.eloping no.el0 cheap0 effecti.e an# safe a#ju.ants ,hich stimulate cellular0 as ,ell
as humoral immunity to *e use# ,ith me#ical an# .eterinary .accines. 'n a##ition0 the recent
unra.elling of numerous immunological path,ays has facilitate# the rational #e.elopment of
ne, a#ju.ants an# allo,e# a *etter un#erstan#ing of the mo#es of action of tra#itional
a#ju.ants.
Mo!e o* action o* immuno#ogica# a!0u'ant"2 "ome 1y"icoc1emica# *acto$" in*#uencing t1e
e**ecti'ity o* o#yac$y#ic a!0u'ant"3
$he a#ju.ant effects of #ifferent polyacrylic pro#ucts an# monomers

,ere teste#. 'nfluen-a
.accine ,as use# as a mo#el antigen.

(##ition of monomers resulte# in a #ecrease in the
anti*o#y

response0 though a#ju.ant acti.ity of the monomers shoul# *e

e1pecte# accor#ing to
some theories on a#ju.ant action. $he

particle si-e of the polymer a#ju.ants pro.e# to *e a .ery
important

parameter for a#ju.ant acti.ity. :articles of B.< to B.I micron

yiel#e# a goo# a#ju.ant
effect0 ,hereas conglomerates or particles

*igger than B.L micron yiel#e# only poor or no
a#ju.ant effects.

$he a#ju.ant effect of B.<3 to B.I3micron particles ,as much

more repro#uci*le
than rat of (l/2"%. (ttention is #ra,n to

the importance of using physiochemically
repro#uci*le materials0

such as polymer particles0 for e1perimental ,ork.

ANIMAL HAN4LING AN4 -EST-AINT
Anima# Han!#ing S5i##"6+$o*e""iona#i"m an! Sa*ety
T $he pu*lic ,atches us to learn ho, to properly han#le animals.
T Being professional means *eing S(FE an# 29M(NE.
T ;oo# animal han#ling skills pre.ent staff from *eing injure#.
T ;oo# animal han#ling skills re#uce stress for the animal.
E&am#e" o* Sa*e Anima# Han!#ing2
T Be a,are of the special stressors for animals in the clinic setting.
T $he clinic is e1tremely chaotic for any animal3there are an incre#i*le num*er of smells
an# other stimuli an# animals are likely to *e confuse# an# #istresse#.
T Many of our patients ha.e li.e# entirely out#oors an# ha.e not *een han#le# or e1amine#
*efore. $hey may not ha.e any e1perience on a leash an# may panic in response.
T E.en the most social animal may e1hi*it aggression to,ar# other animals0 particularly in a
strange en.ironment an# may re#irect to near*y people ,hen o.er3stimulate#.
T Ne.er put your face #irectly into the face of a #og or cat.
T Do not mo.e in *ehin# or cro,# aroun# a #og.
T Concentrate on the animal you are han#ling ,ithout *eing #istracte# *y other acti.ities.
T NE&ER sit on the floor ,hile han#ling+e1amining a #og. 'f the animal *ecomes aggressi.e or
arouse# you ,ill *e una*le to mo.e a,ay or protect yourself an# risk serious facial *ites.
T (l,ays *e prepare# to protect yourself or mo.e a,ay 8uickly in the e.ent an animal *ecomes
aggressi.e une1pecte#ly.
Safe an# effecti.e animal han#ling re8uires a thorough un#erstan#ing of the normal *eha.ior
an#
responses of each species. Belo, is some general information on animal *eha.ior an# han#ling
techni8ues. $here is no su*stitute0 ho,e.er0 for careful o*ser.ation an# e1perience.
Communication
(ny animal e1hi*iting potentially aggressi.e *eha.ior shoul# ha.e a kennel sign C(9$'/N"
poste# to alert others ,ho may *e han#ling the animal. Specific alerts or recommen#ations
shoul# *e ,ritten on the sign an# in the me#ical recor# to pro.i#e staff an# other .olunteers ,ith
as much information as possi*le ,hen han#ling the animal.
-e"t$aint o$ Cont$o#
$he first rule to keep in min# ,hen han#ling any kin# of animal is that the least restraint is often
the *est restraint. $his #oes not mean that you gi.e up your control0 just that you use as little
restraint as necessary ,hile maintaining control of the situation. E.ery animal an# e.ery
situation is #ifferent so as to ,hat metho# ,orks *est in ,hich situation.
Before attempting to restrain an animal you shoul# take a moment to allo, the animal to
*ecome
comforta*le ,ith you:
T Crouch #o,n so that you are on their le.el. Do not sit on the groun# as you ,ill *e una*le to
mo.e a,ay or protect yourself if necessary.
T (.oi# #irect eye contact *ut maintain safe .isual contact ,ith the animal
T $alk in soothing tones. (.oi# high3pitche#0 e1cite# talk.
T $ry patting your leg or the groun#0 motioning the animal to,ar#s you.
TY+ES O, -EST-AINT
7E-BAL -EST-AINT2
Many #ogs kno, some comman#s or can at least recogni-e authority0 e.en if the comman# is
unfamiliar.Comman#s such as S'$0 S$(J0 C/ME0 D/KN0 N/ or e.en 2EE! may *e useful
tools to encourage a #og to cooperate. (lso0 soft 8uiet ,or#s can calm a frightene# animal.
Jelling or screaming shoul# ne.er *e use# as it can cause the animal to *ecome more fearful or
aggressi.e.
+HYSICAL -EST-AINT2 TOOLS AN4 E8UI+MENT
Lea"12 $he most common tool use# to han#le animals in the clinic is the leash. :lace# aroun# a
#ogFs neck it normally controls e.en the largest #og. 'n the e.ent a #og refuses to cooperate ,ith
a leash 3 carry him. Some #ogs ha.e ne.er seen a leash an# ,ill free-e up to the sensation
aroun# a sensiti.e area like the neck. !eashes can *e a*use#5 ne.er #rag or strangle an animal
,ith a leash5 if the animal starts to struggle0 pulling an# jerking a,ay from you0 she is pro*a*ly
not leash traine#. :ause an# let the #og calm #o,n an# try again after reassuring her. Sometimes
a 8uick tug on the leash ,ill encourage a fearful #og to ,alk. 'f the #og refuses to ,alk0 apply a
mu--le if necessary" an# carry her.
Khen han#ling cats0 a leash shoul# *e use# as a *ack3up in the e.ent the cat shoul# *ecome
frightene# an# resist restraint. Make a figure3eight harness *y looping the free en# of a slip lea#
*ack through the metal ring. $he looser loop is place# aroun# the chest *ehin# the catHs front
legs an# the other loop place# aroun# the neck ,ith the metal ring+han#le on top *et,een the
shoul#ers. $his ,ill pre.ent the cat from escaping or injuring someone shoul# she get loose from
your restraint. $he harness shoul# *e put on at intake an# can *e left on the cat throughout their
stay.
E7E-Y anima# %eing t$an"o$te! o$ 1an!#e! in t1e c#inic mu"t AL9AYS :ea$ a "#i6#ea!3
$his inclu#es puppies0 cats an# se#ate# animals. 't is too easy for a frightene# animal to get
loose an# escape. (nimals presente# on leash+collar shoul# *e transferre# to a slip lea# an# the
leash returne# to the client so that it is not lost #uring the animalHs stay.
You$ 1an!2 ( .ery effecti.e form of restraint0 your han#s are sensiti.e to the amount of
pressure that is *eing e1erte# on the animal an# can *e 8uickly mo#ifie# to the situation. 2an#s
can *e use# to gently stroke a #og or to firmly grasp a struggling cat. (lthough han#s can *e the
most .ersatile0 they are also the most .ulnera*le to injury. Recogni-ing ,hen they ,oul# not *e
effecti.e is .ery important.
To:e#"2 ( to,el or *lanket is a .ery useful tool for cats an# small #ogs. ( to,el can *e use# to
#ecrease an animalHs arousal *y co.ering the hea# an# *o#y an# can help protect from sharp
cla,s.
Come6a6#ong o$ cont$o# o#e2 $he control pole is use# to safely han#le e1tremely aggressi.e
#ogs.9se# appropriately it is an effecti.e tool. 'nappropriate or unskille# use can cause serious
injury to the animal. $he control pole may further #istress an upset animal an# shoul# only *e
use# ,hen the han#ler or otherFs safety is genuinely threatene#. &olunteers are N/$ to use the
control pole unassiste#. 'f an animal is aggressi.e enough to ,arrant the use of a control pole an
e1perience# staff mem*er shoul# *e consulte# for assistance as the animal ,ill also *e e.aluate#
for chemical restraint options.
Net"2 $he net is the primary tool use# to han#le fractious cats or ,il#life. 't allo,s for the safe
han#ling an# transfer of e.en the most aggressi.e small mammal. Effecti.e use of the net
re8uires some training
an# practice. 'f you nee# to han#le a feral or fractious cat ask for assistance from a staff mem*er.
Mu;;#e"2 Mu--les are use# ,hen a snappy or potentially aggressi.e #og must *e han#le#. $here
are nylon mu--les an# plastic *asket a.aila*le. ( leash or strip of rolle# gau-e can *e use# as a
temporary mu--le. Because #ogs often try to remo.e a mu--le0 it is important that the mu--le *e
place# securely.
( ,eak or poorly ma#e mu--le may lea# to a false sense of security an# the possi*ility of *eing
*itten. E.en ,ith a securely place# mu--le0 appropriate han#ling must *e use# to pre.ent injury
from an animal ,ho resists.Mu--les #esigne# for cats e1ten# up to co.er the eyes0 re#ucing
.isual stimulation. For some cats these can *e .ery useful for calming the animal an# helping to
protect the han#ler from injury0
4$ug"2 For animals ,ho are too aggressi.e or stresse# to han#le safely for proce#ures0 se#ation
an#+or general anesthesia may *e necessary to allo, treatment. 'f you are una*le to han#le an
animal0 notify a staff mem*er to #etermine ,hether se#ation is appropriate. Khen recei.ing an
animal for surgery ,ho e1hi*its #ifficult or aggressi.e *eha.ior consult the (nesthesia
!ea# prior to kenneling the animal as ,e may opt to a#minister a pre3anesthetic se#ati.e
imme#iately
an# e1pe#ite the surgery process to minimi-e the animalHs time in the clinic.
C$e!o2 Ne'e$ Let Go3
$he place ,here correct use of restraint is the most critical is ,hen t,o people are han#ling the
animal.$his coul# *e to perform a physical e1am0 a#minister anesthetic or to gi.e me#ications.
$he 4hol#er4 is the person ,hose jo* it is to restrain the animal in such a ,ay that the proce#ure
can *e accomplishe# ,ith the least amount of stress to *oth han#lers an# animal. $he specific
amount of restraint use# to control the animal is the key to safety for the han#lers an# comfort
for the animal. $oo much restraint can cause the animal to fight *ack0 too little restraint can
result in the han#ler or others *eing injure# or in the animal escaping.
Restraint and Handling of Animals
General Principles
The use of proper restraint and handling techniques reduces stress to animals and also to the
researcher. Handling stress represents an experimental variable and should be minimized whenever
possible. Animals can inflict serious injuries to humans and to themselves as a result of improper
handling.
Animals experience stress as a result of shipping. All large animals must be allowed to acclimate
to the facility for three days. During this time they may not be experimentally manipulated.
Acclimation periods of up to one wee are recommended for all animals.
!f a study will involve significant handling of animals it is recommended that the animals be
acclimated to the handling. "rior to experimental manipulation# handle the animal on a regular
basis in a non$threatening situation# e.g. weighing# petting# giving food treats. %ost animals# even
rodents will respond positively to handling and will learn to recognize individuals.
Handle animals gently. Do not mae loud noises or sudden movements that may startle them.
Handle animals firmly. The animal will struggle more if it sees a chance to escape.
&se an assistant whenever possible.
&se restraint devices to assist when appropriate.
'hemical restraint should be considered for any prolonged or potentially painful procedure.
Handling Methods
The methods described below will assist with performing basic manipulations. Alternate techniques may
be needed for special procedures. %ost of these methods are also demonstrated in video tapes available
to investigator. (or other information on animal handling or for individual training# contact )A) at *+,$
-.//. An excellent website containing laboratory biomethodology for rodents and rabbits is also available
with descriptions and pictures of drug administration# blood collection and sex determination.
Needle Re-Use Policy
The use of a new sterile needle and syringe for each animal when giving parenteral injections
0intraperitoneal# subcutaneous# intravenous# intramuscular# etc.1 is the recommended best practice to
prevent the horizontal transfer of contamination between animals. However# the !A'&' recognizes that
there are some instances where it may be justified to use the same needle and syringe for multiple
animals# usually in rodents. !n those instances the "rincipal !nvestigator must provide justification to the
!A'&' and must adhere to the following guidelines. &se of the same needle and syringe may be
permitted with justification on animals housed in the same cage. The needle must be assessed for
continued sharpness and the presence of barbing or burring of the tip between animals. !f dullness or
needle deterioration is found# a new needle must be used.
MICE
Tail restraint# as described below is adequate for examining
animals and transfering them to another cage.
These methods may be used to perform minor# non$
painful procedures such as injections or ear tagging.
RATS may be handled by the tail# with precautions similar to
those used for mice# with emphasis on only grasping the tail
base. Holding the tail distal to the base can result in a de$
gloving injury to the tail that will require surgical repair or
euthanasia.
This method should be used to restrain a rat for
injections and other minor procedures.
HAMSTERS
2ecause hamsters do not have tails# they must be grasped
firmly by the loose sin of its bac# or handled in a manner
similar to the rat.
GUINEA PIGS rarely bite# but are very easily
frightened and will vocalize and squirm to avoid
restraint. The hind limbs must be supported at all
times to prevent the animal from injuring its bac.
RABBITS are very susceptible to lumbar spinal luxation#
resulting in paralysis. !t is necessary to support the animal3s
hindquarter at all times. Although rabbits seldom bite# they
can inflict painful scratches with their hind legs. 4ne way of
lifting a rabbit is by grasping the sin over the shoulder with
one hand and gently lifting it with the other arm cradling the
body# the head nestled in the croo of your arm. )abbits must
never by lifted by the ears.
CATS are often cooperative enough to be restrained on a
table by the loose sin at the bac of the nec and hips# or
with one hand restraining the body and the other restraining
the head. A fractious cat may have to be wrapped in a heavy
towel for restraint with any needed limbs carefully withdrawn
for treatment.
!GS
A slip lead is highly recommended for woring with dogs. A
dog should always be carried with proper support. The dog
can be restrained in lateral recumbancy or in a sitting position
for injections and minor procedures. (or venipuncture# the
handler can restrain the dog on a table with one arm around
its nec. The other hand is then free to restrain the body if
necessary or to occlude the vein for the person with the
syringe. A shy or fearful dog may need extra time spent with it
to mae it more comfortable. %oving slowly and speaing
quietly will help to prevent alarming the animal.

An intractable dog may need to be muzzled. A
commercial muzzle may be purchased# or a gauze
muzzle may used as described below.
"ills are easily administered to most dogs if the
proper technique is used.
N!NHUMAN PRIMATES# no matter how small# can be
dangerous. 'hemical immobilization with etamine is
nolrmally used. !njections can be given to a confined animal
with the help of a squeeze cage.
Safety:
Absolute requirements for handling of nonhuman
primates include attending a training module given
by )A) 0contact *+,$-.// to schedule1# and wearing
"hysical restraint of a conscious animal should only be
attempted by trained# experienced personnel. Animals may
be pole and collar trained if they will be handled frequently.
Tether systems are recommended if animals must be
administered drugs or if blood must be collected frequently.
appropriate protective clothing. !n addition# nonhuman
primate users should be familiar with procedures to
follow in case of a bite or scratch and the location of
bite its.
!f a nonhuman primate has escaped# close all doors
and contact )A) at *+,$-.//. The animal may be
recaptured using a net or a dart gun.
G!ATS" SHEEP and CA#$ES
)estrain against a wall or in a corner by placing a
nee firmly in the flan.
)estrain for blood collection by bacing the animal
into a corner and straddling them at the shoulder and
firmly restraining the head and nec.
&se a halter over their head and face.
A sheep can be held for bleeding# shearing or hoof
trimming by sitting the animal up on its hind end#
leaning bac against the restrainer.
(or long term restraint of sheep in the laboratory# a
canvas sling and rac is available from several
commercial suppliers. Animals are easily acclimated
to such slings# and can be comfortable and relaxed
enough to fall asleep in them.
Additional references on handling of agricultural
animals is available from the &5DA.
Temple 6randin3s 7ebsite on #o%-Stress Handling
o& 'ar( Ani(als
RESTRAINT AN HAN#ING !' S)INE
By r* +ac, Risdahl
Additional information on restraint of# and blood collection from# swine may be found on the &'Davis website.
http899ehs.ucdavis.edu9animal9vet:care9training9"ig.cfm
"igs in general are friendly and docile but will react severely to poor handling or a stressful environment. "igs can
be very vocal. !f pigs are chronically stressed they will become sittish and fearful. Handling and restraint in pigs
relies greatly on treating the pigs in a humane manner. The benefits of treating pigs well include reducing
apprehension# fear and stress in the pigs. There are several levels of restraint and handling# from touching and
coaxing a pig to restraining a pig for chronic procedures.
Touch is a very important aid to good husbandry.
Ani(al-H-(an Contact
7hen approaching a pig be sure it is made aware of your presence. !f pigs
are startled they may cause injury to themselves or others in the pen. The
best way to mae pigs aware of your presence is to use your voice. !t is
important to use a soft soothing voice and not angry# loud# high pitched
tone of voice which might startle or stress the animal. "igs quicly learn to
recognize voices# especially if they are associated with food. As pigs become familiar with handlers# the sound of a
familiar voice is often calming to the animal. !t is important to use touch when developing a rapport with pigs. This
applies especially to the researcher who must collect frequent samples or data from pigs. As with voice# gentle
petting and hand contact should be associated with feeding time or treats and the pig will become aware of the
person in the vicinity and become adjusted to that persons presence. "robably one of the best forms of restraint in
pigs is the use of food. "igs are highly oriented to food and if they are comfortable with the handler will most often
stand and eat while minor procedures and examinations are being performed on them. 4ne can often flush
catheters# give injections# treat minor wounds and tae temperatures while pigs eat. The use of all three procedures
$ voice# touch# and food# will be the best investment in reducing stress among research swine and will ultimately
reward the researcher with a happy stress free subject.
The giving of food is one of the most effective forms of basic restraint in the pig.
Pic,ing Up Pigs
"igs best tolerate being piced up in a ;horizontal; fashion oriented to the
ground. "igs should not be piced up by the legs or held upside down as this
will stress the animal and you will loose their trust. &sually only smaller
animals may be piced up while larger animals 0<=>$,/ g1 must be moved
by alternative means. 5maller pigs may be easily piced up with their body supported while their legs hang. To
perform the procedure in larger pigs place one arm under the chest cranial to the thoracic limbs and the other arm
cranial to the pelvic limbs under the abdomen picing up the pig in a ;scooping; fashion. Alternatively the arm may
be placed caudally just above the pig3s hoc# hence supporting the animal by the pelvis rather than the abdomen. All
handlers must beware to lift with legs and not bac as injury can easily result $ pigs are usually heavier than they
appear? Always avoid picing pigs up by one leg or by the ears as injury may result?
Mo.ing Pigs
The small board used to apply pressure to the side of a pig.
"igs are best moved in a metal 0box style1 transport designed for use with large
animals. At times this is not possible and pigs must be waled to their destination.
7hen moving a pig always remember pigs will move away from walls toward
openings. This is an advantage since one can use a ;hog board; to simulate walls.
The board is fashioned with a handle so that one can place it to the side# rear or
front of the pig to direct them. @xcessive force should not be needed to move a pig
and is mostly counterproductive as pigs will become excited and belligerent. !t
should be remembered pigs will refuse to move if the place you wish them to go is
dar 0e.g. from daylight into a dar room1. 5ometimes pigs may be coaxed with
food along with the use of the board. 7hen pigs are unruly and where control is
needed# pigs may be tethered in a harness and controlled by ;holder; so that the pig does not run away. 4ften the
use of the hog board may be used to stop pig and slow them down if they are moving too rapidly. The board may
also be used to restrain a pig in a corner while minor procedures are performed. The size of the board varies
depending on the size of pigs used and application. !n general if the board is at least as tall as the pig and +9= to
about as long as the pig it will usually suffice.
Sling
5everal designs for slings to restrain pigs have been described. The most commonly used is
that described by "anepinto et al .-A=. Here the pig is placed in a hammoc with four holes
for the limbs. The hammoc is supported by a metal frame. These are available in free
standing or winch styles 0so larger pigs may be raised by winch1. The pig is placed in ventral
recumbency in the sling with its limbs tied loosely to the frame. !t has been our experience
that this form of restraint requires some degree of training for pigs to acclimate to. !n general
most pigs will become stressed the first several times they are placed in the sling. "ositive
reinforcement 0treats# petting1 and repetition usually calms them down so that they may be
restrained for extended periods in the sling. 7e have generally used a training period of two
wees prior to experimental procedures with a minimum of =/ min.9day in the sling. !n our
experience one or two hours is about the most a pig will tolerate.
Accli(ation and Sociali/ation
!t should be remembered that pigs are social animals and have a rigid dominance hierarchy. !f animals are group
housed they will generally fight to establish dominance for the first +,$,A hours. Dominance in pigs is almost directly
related to size. The largest animals are dominant and smallest are submissive. 2e sure to match weights as close
as possible when introducing new pigs to each other. 5maller pigs may be injured by larger pigs. 2e sure to monitor
pigs for the introduction period so that they do not cause major injury to each other $ they will fight. Always
remember that newly arrived pigs are stressed from transport. Do not initiate experimental procedures in the first
few days of arrival. This is just common sense as immune function and physiologic parameters are often altered by
stress. 7e lie to see an acclimation period of two wees so that pigs may adapt to their new environment and
establish rapport with handlers.
r-g ad(inistration
A butterfly needle can be attached to a syringe for administering injections to swine# allowing them to move
during the injection without displacing the needle.
!ntravenous injections may be given in the ear veins.
4ral drugs may be administered ground or whole mixed with a food treat.
"ills can be administered orally as with dogs. However# the handler must be sure to get the pill all the way
behind the tongue and must avoid being bitten.
5ome drugs may be administered rectally. A literature review should be performed for the drug in question
prior to attempting this.
'n#,elling central .enous catheters are recommen#e# if animals ,ill *e recei.ing #rugs on a
regular *asis.
Restraint evices
)estraint devices such as rabbit or rodent restrainers# swine slings or money chairs are useful for
certain non$painful procedures. However# certain guidelines should be followed when using these
devices.
Animals should be adapted to the restraint devices. This means that for long$term restraint 0i.e.
more than an hour1# it is advisable to ;train; the animal to the device by placing it into the device
for successively longer intervals until the maximum time of restraint can be achieved without
causing distress to the animal.
Animals in a restraint device be regularly monitored. This means not leaving the area for long
intervals unless someone else is available to monitor the animal. Animals have an uncanny
ability to attempt escape from devices# if they don3t succeed completely# they may end up with a
limb or their head entrapped. This could result in ischemia or hypoxia.
Animals should have access to food or water at appropriate intervals# even when restrained#
unless doing so would interfere with the goals of the experiment. (ood or water should be
offered twice daily. (or rabbits and rodents# water should be offered more frequently.
Animals should be released from restraint devices at least daily and allowed unrestrained activity
to prevent muscle atrophy and sin necrosis# unless this interferes with achieving the
experimental goals and is documented in an approved !A'&' protocol.
UNIT II ANTIBO4IES < IMMUNO4IAGNOSIS
Monoc#ona# anti%o!ie"
A gene$a# $e$e"entation o* t1e met1o!" u"e! to $o!uce monoc#ona# anti%o!ie"3
Monoc#ona# anti%o!ie" mA% or moA%" are monospecific anti*o#ies that are the same *ecause
they are ma#e *y i#entical immune cells that are all clones of a uni8ue parent cell.
;i.en almost any su*stance0 it is possi*le to create monoclonal anti*o#ies that specifically *in#
to that su*stance5 they can then ser.e to #etect or purify that su*stance. $his has *ecome an
important tool in *iochemistry0 molecular *iology an# me#icine. Khen use# as me#ications0 the
non3proprietary #rug name en#s in -mab.
4i"co'e$y
$he i#ea of a 4magic *ullet4 ,as first propose# *y :aul Ehrlich0 ,ho0 at the *eginning of the
IBth century0 postulate# that0 if a compoun# coul# *e ma#e that selecti.ely targete# a #isease3
causing organism0 then a to1in for that organism coul# *e #eli.ere# along ,ith the agent of
selecti.ity. 2e an# Ulie Metchnikoff recei.e# the <>B= No*el :ri-e for :hysiology or Me#icine
for this ,ork0 ,hich le# to an effecti.e syphilis treatment *y <><B.
'n the <>?Bs0 the B3cell cancer multiple myeloma ,as kno,n0 an# it ,as un#erstoo# that these
cancerous B3cells all pro#uce a single type of anti*o#y a paraprotein". $his ,as use# to stu#y
the structure of anti*o#ies0 *ut it ,as not yet possi*le to pro#uce i#entical anti*o#ies specific to
a gi.en antigen.
:ro#uction of monoclonal anti*o#ies in.ol.ing humanAmouse hy*ri# cells ,as #escri*e# *y
Verrol# Sch,a*er in <>?%an# remains ,i#ely cite# among those using human3#eri.e#
hy*ri#omas0 *ut claims to priority ha.e *een contro.ersial. ( science history paper on the
su*ject ga.e some cre#it to Sch,a*er for in.enting a techni8ue that ,as ,i#ely cite#0 *ut
stoppe# short of suggesting that he ha# *een cheate#. $he in.ention ,as concei.e# *y ;eorge
:iec-enik0 ,ith Vohn Se#at0 Eli-a*eth Black*urnFs hus*an#0 as a ,itness an# re#uce# to practice
*y Cotton an# Milstein0 an# then *y Nohler an# Milstein. ;eorges NDhler0 CWsar Milstein0 an#
Niels Naj Verne in <>?L5 ,ho share# the No*el :ri-e in :hysiology or Me#icine in <>=@ for the
#isco.ery. $he key i#ea ,as to use a line of myeloma cells that ha# lost their a*ility to secrete
anti*o#ies0 come up ,ith a techni8ue to fuse these cells ,ith healthy anti*o#y3pro#ucing B3
cells0 an# *e a*le to select for the successfully fuse# cells.
'n <>==0 ;reg Kinter an# his team pioneere# the techni8ues to humani-e monoclonal anti*o#ies0
remo.ing the reactions that many monoclonal anti*o#ies cause# in some patients.
+$o!uction
Monoclonal anti*o#ies can *e gro,n in unlimite# 8uantities in the *ottles sho,n in this picture.
$echnician han#3filling ,ells ,ith a li8ui# for a research test. $his test in.ol.es preparation of
cultures in ,hich hy*ri#s are gro,n in large 8uantities to pro#uce #esire# anti*o#y. $his is
effecte# *y fusing myeloma cell an# mouse lymphocyte to form a hy*ri# cell hy*ri#oma".
Hy%$i!oma ce## $o!uction
Monoclonal anti*o#ies are typically ma#e *y fusing myeloma cells ,ith the spleen cells from a
mouse that has *een immuni-e# ,ith the #esire# antigen. 2o,e.er0 recent a#.ances ha.e
allo,e# the use of ra**it B3cells. :olyethylene glycol is use# to fuse a#jacent plasma
mem*ranes0 *ut the success rate is lo, so a selecti.e me#ium in ,hich only fuse# cells can
gro, is use#. $his is *ecause myeloma cells ha.e lost the a*ility to synthesi-e hypo1anthine3
guanine3phosphori*osyl transferase 2;:R$"0 an en-yme necessary for the sal.age synthesis of
nucleic aci#s. $he a*sence of 2;:R$ is not a pro*lem for these cells unless the #e no.o purine
synthesis path,ay is also #isrupte#. By e1posing cells to aminopterin a folic aci# analogue0
,hich inhi*its #ihy#rofolate re#uctase0 D2FR"0 they are una*le to use the #e no.o path,ay an#
*ecome fully au1otrophic for nucleic aci#s re8uiring supplementation to sur.i.e.
$he selecti.e culture me#ium is calle# 2($ me#ium *ecause it contains hypo1anthine0
aminopterin0 an# thymi#ine. $his me#ium is selecti.e for fuse# hy*ri#oma" cells. 9nfuse#
myeloma cells cannot gro, *ecause they lack 2;:R$0 an# thus cannot replicate their DN(.
9nfuse# spleen cells cannot gro, in#efinitely *ecause of their limite# life span. /nly fuse#
hy*ri# cells0 referre# to as hy*ri#omas0 are a*le to gro, in#efinitely in the me#ia *ecause the
spleen cell partner supplies 2;:R$ an# the myeloma partner has traits that make it immortal as
it is a cancer cell".$his mi1ture of cells is then #ilute# an# clones are gro,n from single parent
cells on microtitre ,ells. $he anti*o#ies secrete# *y the #ifferent clones are then assaye# for
their a*ility to *in# to the antigen ,ith a test such as E!'S( or (ntigen Microarray (ssay" or
immuno3#ot *lot. $he most pro#ucti.e an# sta*le clone is then selecte# for future use.
$he hy*ri#omas can *e gro,n in#efinitely in a suita*le cell culture me#ia0 or they can *e
injecte# in mice in the peritoneal ca.ity0 the gut"0 they pro#uce tumors containing an anti*o#y3
rich flui# calle# ascites flui#. $he me#ium must *e enriche# #uring selection to further fa.our
hy*ri#oma gro,th. $his can *e achie.e# *y the use of a layer of fee#er fi*rocyte cells or
supplement me#ium such as *riclone. Culture3me#ium con#itione# *y macrophages can also *e
use#. :ro#uction in cell culture is usually preferre# as the ascites techni8ue is painful to the
animal an# if replacement techni8ues e1ist0 this metho# is consi#ere# unethical.
+u$i*ication o* monoc#ona# anti%o!ie"
(fter o*taining either a me#ia sample of culture# hy*ri#omas or a sample of ascites flui#0 the
#esire# anti*o#ies must *e e1tracte#. $he contaminants in the cell culture sample ,oul# consist
primarily of me#ia components such as gro,th factors0 hormones0 an# transferrins. 'n contrast0
the in .i.o sample is likely to ha.e host anti*o#ies0 proteases0 nucleases0 nucleic aci#s0 an#
.iruses. 'n *oth cases0 other secretions *y the hy*ri#omas such as cytokines may *e present.
$here may also *e *acterial contamination an#0 as a result0 en#oto1ins that are secrete# *y the
*acteria. Depen#ing on the comple1ity of the me#ia re8uire# in cell culture0 an# thus the
contaminants in 8uestion0 one metho# in .i.o or in .itro" may *e prefera*le to the other.
$he sample is first con#itione#0 or prepare# for purification. Cells0 cell #e*ris0 lipi#s0 an# clotte#
material are first remo.e#0 typically *y centrifugation follo,e# *y filtration ,ith a B.@L Qm
filter. $hese large particles can cause a phenomenon calle# mem*rane fouling in later
purification steps. 'n a##ition0 the concentration of pro#uct in the sample may not *e sufficient0
especially in cases ,here the #esire# anti*o#y is one pro#uce# *y a lo,3secreting cell line. $he
sample is therefore con#ense# *y ultrafiltration or #ialysis.
Most of the charge# impurities are usually anions such as nucleic aci#s an# en#oto1ins. $hese
are often separate# *y ion e1change chromatography0 using columns such as the :ro:ac KCX3
<B0 no, regar#e# as the gol# stan#ar# for anti*o#y analysis. Either cation e1change
chromatography is use# at a lo, enough p2 that the #esire# anti*o#y *in#s to the column ,hile
anions flo, through0 or anion e1change chromatography is use# at a high enough p2 that the
#esire# anti*o#y flo,s through the column ,hile anions *in# to it. &arious proteins can also *e
separate# out along ,ith the anions *ase# on their isoelectric point p'". For e1ample0 al*umin
has a p' of @.=0 ,hich is significantly lo,er than that of most monoclonal anti*o#ies0 ,hich ha.e
a p' of E.<. 'n other ,or#s0 at a gi.en p20 the a.erage charge of al*umin molecules is likely to
*e more negati.e. $ransferrin0 on the other han#0 has a p' of L.>0 so it cannot easily *e separate#
out *y this metho#. ( #ifference in p' of at least < is necessary for a goo# separation.
$ransferrin can instea# *e remo.e# *y si-e e1clusion chromatography. $he a#.antage of this
purification metho# is that it is one of the more relia*le chromatography techni8ues. Since ,e
are #ealing ,ith proteins0 properties such as charge an# affinity are not consistent an# .ary ,ith
p2 as molecules are protonate# an# #eprotonate#0 ,hile si-e stays relati.ely constant.
Nonetheless0 it has #ra,*acks such as lo, resolution0 lo, capacity an# lo, elution times.
( much 8uicker0 single3step metho# of separation is :rotein (+; affinity chromatography. $he
anti*o#y selecti.ely *in#s to :rotein (+;0 so a high le.el of purity generally Y=BZ" is o*taine#.
2o,e.er0 this metho# may *e pro*lematic for anti*o#ies that are easily #amage#0 as harsh
con#itions are generally use#. ( lo, p2 can *reak the *on#s to remo.e the anti*o#y from the
column. 'n a##ition to possi*ly affecting the pro#uct0 lo, p2 can cause :rotein (+; itself to
leak off the column an# appear in the elute# sample. ;entle elution *uffer systems that employ
high salt concentrations are also a.aila*le to a.oi# e1posing sensiti.e anti*o#ies to lo, p2. Cost
is also an important consi#eration ,ith this metho# *ecause immo*ili-e# :rotein (+; is a more
e1pensi.e resin.
$o achie.e ma1imum purity in a single step0 affinity purification can *e performe#0 using the
antigen to pro.i#e e18uisite specificity for the anti*o#y. 'n this metho#0 the antigen use# to
generate the anti*o#y is co.alently attache# to an agarose support. 'f the antigen is a pepti#e0 it
is commonly synthesi-e# ,ith a terminal cysteine0 ,hich allo,s selecti.e attachment to a carrier
protein0 such as N!2 #uring #e.elopment an# to the support for purification. $he anti*o#y3
containing me#ia is then incu*ate# ,ith the immo*ili-e# antigen0 either in *atch or as the
anti*o#y is passe# through a column0 ,here it selecti.ely *in#s an# can *e retaine# ,hile
impurities are ,ashe# a,ay. (n elution ,ith a lo, p2 *uffer or a more gentle0 high salt elution
*uffer is then use# to reco.er purifie# anti*o#y from the support.
$o further select for anti*o#ies0 the anti*o#ies can *e precipitate# out using so#ium sulfate or
ammonium sulfate. (nti*o#ies precipitate at lo, concentrations of the salt0 ,hile most other
proteins precipitate at higher concentrations. $he appropriate le.el of salt is a##e# in or#er to
achie.e the *est separation. E1cess salt must then *e remo.e# *y a #esalting metho# such as
#ialysis.
$he final purity can *e analy-e# using a chromatogram. (ny impurities ,ill pro#uce peaks0 an#
the .olume un#er the peak in#icates the amount of the impurity. (lternati.ely0 gel
electrophoresis an# capillary electrophoresis can *e carrie# out. 'mpurities ,ill pro#uce *an#s of
.arying intensity0 #epen#ing on ho, much of the impurity is present.
-ecom%inant
$he pro#uction of recom*inant monoclonal anti*o#ies in.ol.es technologies0 referre# to as
repertoire cloning or phage display/yeast display. Recom*inant anti*o#y engineering in.ol.es
the use of .iruses or yeast to create anti*o#ies0 rather than mice. $hese techni8ues rely on rapi#
cloning of immunoglo*ulin gene segments to create li*raries of anti*o#ies ,ith slightly #ifferent
amino aci# se8uences from ,hich anti*o#ies ,ith #esire# specificities can *e selecte#. $he
phage anti*o#y li*raries are a .ariant of the phage antigen li*raries first in.ente# *y ;eorge
:iec-enik $hese techni8ues can *e use# to enhance the specificity ,ith ,hich anti*o#ies
recogni-e antigens0 their sta*ility in .arious en.ironmental con#itions0 their therapeutic efficacy0
an# their #etecta*ility in #iagnostic applications. Fermentation cham*ers ha.e *een use# to
pro#uce these anti*o#ies on a large scale.
C1ime$ic anti%o!ie"
Early on0 a major pro*lem for the therapeutic use of monoclonal anti*o#ies in me#icine ,as that
initial metho#s use# to pro#uce them yiel#e# mouse0 not human anti*o#ies. Khile structurally
similar#ifferences *et,een the t,o sufficient to in.oke an immune response occurre# ,hen
murine monoclonal anti*o#ies ,ere injecte# into humans an# resulte# in their rapi# remo.al
from the *loo#0 systemic inflammatory effects0 an# the pro#uction of human anti3mouse
anti*o#ies 2(M(".
'n an effort to o.ercome this o*stacle0 approaches using recom*inant DN( ha.e *een e1plore#
since the late <>=Bs. 'n one approach0 mouse DN( enco#ing the *in#ing portion of a
monoclonal anti*o#y ,as merge# ,ith human anti*o#y3pro#ucing DN( in li.ing cells0 an# the
e1pression of this chimeric DN( through cell culture yiel#e# half3mouse0 half3human
monoclonal anti*o#y. For this pro#uct0 the #escripti.e terms 4chimeric4 an# 4humanise#4
monoclonal anti*o#y ha.e *een use# to reflect the amount of human DN( use# in the
recom*inant process.
=,u##y= 1uman monoc#ona# anti%o!ie"
E.er since the #isco.ery that monoclonal anti*o#ies coul# *e generate# in3.itro0 scientists ha.e
targete# the creation of FfullyF human anti*o#ies to a.oi# some of the si#e effects of humanise#
an# chimeric anti*o#ies. $,o successful approaches ,ere i#entifie# 3 phage #isplay3generate#
anti*o#ies an# mice genetically engineere# to pro#uce more human3like anti*o#ies.
/ne of the most successful commercial organisations *ehin# therapeutic monoclonal anti*o#ies
,as Cam*ri#ge (nti*o#y $echnology C($". Scientists at C($ #emonstrate# that phage #isplay
coul# *e use# such that .aria*le anti*o#y #omains coul# *e e1presse# on filamentous phage
anti*o#ies. $his ,as reporte# in a key Nature pu*lication.
C($ #e.elope# their #isplay technologies further into se.eral0 patente# anti*o#y
#isco.ery+functional genomics tools0 ,hich ,ere name# :ro1imol
$M
an# :ro(*
$M
. :ro(* ,as
announce# in Decem*er <>>? an# in.ol.e# highthroughput screening of anti*o#y li*raries
against #isease# an# non3#isease# tissue0 ,hilst :ro1imol use# a free ra#ical en-ymatic reaction
to la*el molecules in pro1imity to a gi.en protein
;enetically engineere# mice0 so calle# transgenic mice0 can *e mo#ifie# to pro#uce human
anti*o#ies0 an# this has *een e1ploite# *y a num*er of commercial organisations:
Me#are1 3 ,ho market their 9ltiMa* platform
(*geni1 3 ,ho markete# their Xenomouse technology. (*geni1 ,ere ac8uire# in (pril
IBBE *y (mgen.
RegeneronFs &eloc'mmune technology.
Monoclonal anti*o#ies ha.e *een generate# an# appro.e# to treat: cancer0 car#io.ascular
#isease0 inflammatory #iseases0 macular #egeneration0 transplant rejection0 multiple sclerosis0
an# .iral infection see monoclonal anti*o#y therapy".
'n (ugust IBBE the :harmaceutical Research an# Manufacturers of (merica reporte# that 9.S.
companies ha# <EB #ifferent monoclonal anti*o#ies in clinical trials or a,aiting appro.al *y the
Foo# an# Drug (#ministration.
A#ication"
4iagno"tic te"t"
/nce monoclonal anti*o#ies for a gi.en su*stance ha.e *een pro#uce#0 they can *e use# to
#etect the presence of this su*stance. $he Kestern *lot test an# immuno #ot *lot tests #etect the
protein on a mem*rane. $hey are also .ery useful in immunohistochemistry0 ,hich #etect
antigen in fi1e# tissue sections an# immunofluorescence test0 ,hich #etect the su*stance in a
fro-en tissue section or in li.e cells.
T1e$aeutic t$eatment
Monoclonal anti*o#y therapy for Cance$ t$eatment
/ne possi*le treatment for cancer in.ol.es monoclonal anti*o#ies that *in# only to cancer cell3
specific antigens an# in#uce an immunological response against the target cancer cell. Such
m(* coul# also *e mo#ifie# for #eli.ery of a to1in0 ra#ioisotope0 cytokine or other acti.e
conjugate5 it is also possi*le to #esign *ispecific anti*o#ies that can *in# ,ith their Fa* regions
*oth to target antigen an# to a conjugate or effector cell. 'n fact0 e.ery intact anti*o#y can *in#
to cell receptors or other proteins ,ith its Fc region.
Monoc#ona# anti%o!ie" *o$ cance$3 (DE:$0 anti*o#y #irecte# en-yme pro#rug therapy5
(DCC0 anti*o#y #epen#ent cell3me#iate# cytoto1icity5 CDC0 complement #epen#ent
cytoto1icity5 M(*0 monoclonal anti*o#y5 scF.0 single3chain F. fragment.
$he illustration *elo, sho,s all these possi*ilities:
M(*s appro.e# *y the FD( inclu#e
Be.aci-uma*
Cetu1ima*
:anitumuma*
$ra-tu-uma*
:ertu-uma*
Autoimmune !i"ea"e"
Monoclonal anti*o#ies use# for autoimmune #iseases inclu#e infli1ima* an# a#alimuma*0
,hich are effecti.e in rheumatoi# arthritis0 CrohnFs #isease an# ulcerati.e Colitis *y their a*ility
to *in# to an# inhi*it $NF3[.
6I%7
Basili1ima* an# #acli-uma* inhi*it '!3I on acti.ate# $ cells
an# there*y help pre.enting acute rejection of ki#ney transplants.
6I%7
/mali-uma* inhi*its human
immunoglo*ulin E 'gE" an# is useful in mo#erate3to3se.ere allergic asthma.
E&am#e"
Belo, are e1amples of clinically important monoclonal anti*o#ies.
Main
catego$y
Tye A#ication Mec1ani"m>Ta$get Mo!e
Anti6
in*#ammato$y
in*#i&ima%
rheumatoi#
arthritis
CrohnFs #isease
9lcerati.e Colitis
inhi*its $NF3[ chimeric
a!a#imuma%
rheumatoi#
inhi*its $NF3[ human
arthritis
CrohnFs #isease
9lcerati.e Colitis
etane$cet
rheumatoi#
arthritis
Contains #ecoy $NF
receptor
fusion
protein
%a"i#i&ima%
(cute rejection of
ki#ney transplants
inhi*its '!3I on acti.ate#
$ cells
chimeric
!ac#i;uma%
(cute rejection of
ki#ney transplants
inhi*its '!3I on acti.ate#
$ cells
humani-e#
oma#i;uma%
mo#erate3to3
se.ere allergic
asthma
inhi*its human
immunoglo*ulin E 'gE"
humani-e#
Anti6cance$
gemtu;uma%
relapse# acute
myeloi#
leukaemia
targets myeloi# cell
surface antigen CD%% on
leukemia cells
humani-e#
a#emtu;uma% B cell leukemia
targets an antigen CDLI
on $3 an# B3
lymphocytes
humani-e#
$itu&ima%
non32o#gkinFs
lymphoma
targets phosphoprotein
CDIB on B lymphocytes
chimeric
t$a"tu;uma%
*reast cancer ,ith
2ERI+neu
o.ere1pression
targets the 2ERI+neu
er*BI" receptor
humani-e#
nimotu;uma%
(ppro.e# in
s8uamous cell
carcinomas0
;lioma
Clinical trials for
other in#ications
un#er,ay
E;FR inhi*itor 2umani-e#
cetu&ima%
(ppro.e# in
s8uamous cell
carcinomas0
colorectal
carcinoma
E;FR inhi*itor Chimeric
%e'aci;uma%
(nti3angiogenic
cancer therapy
inhi*its &E;F humani-e#
Ot1e$ a#i'i;uma%
6
RS& infections in
inhi*its an RS& fusion
F" protein
humani-e#
chil#ren
a%ci&ima%
:re.ent
coagulation in
coronary
angioplasty
inhi*its the receptor
;p''*+'''a on platelets
chimeric
-ai! C1a$acte$i;ation o* Monoc#ona# Anti%o!ie" u"ing t1e +ie;oe#ect$ic
Immuno"en"o$
Monoclonal anti*o#ies ,ith specificity against the Francisella tularensis outer
lipopolysacchari#e !:S" mem*rane ,ere prepare# an# characteri-e# using the pie-oelectric
immunosensor ,ith immo*ili-e# !:S antigen from F. tularensis. Signals o*taine# *y the
immunosensor ,ere compare# ,ith E!'S( an# similar sensiti.ity ,as notice#. Signal of
negati.e controls o*taine# using the *iosensor ,as *elo, B.LZ of the signal o*taine# for the
selecte# specific anti*o#y clone @2%B>D%. $esting of cross reacti.ity *ase# on the sensors
,ith immo*ili-e# !:S from Escherichia coli an# Bacillus subtilis confirme# selecti.ity of
this anti*o#y. Furthermore0 the @2%B>D% anti*o#y ,as successfully isotypi-e# as 'gM using
the pie-oelectric sensors ,ith secon#ary anti*o#ies. Ninetics parameters of anti*o#y ,ere
e.aluate# in the flo,3through arrangement. $he kinetic rate constants for the anti*o#y
@2%B>D% ,ere ka C I.%< \ B.IB"]<BL l mol3<s3< association" an# k# C B.BB<B \B.BBBEI" s3
<#issociation" in#icating .ery goo# affinity to the !:S antigen.
C1a$acte$i;ation o* Monoc#ona# Anti%o!y +$o!uct"
Characteri-ation tests
A :ro.i#e #etaile# information on the molecule+pro#uct
A Re8uirement for Reference Stan#ar# characteri-ation
A Re8uire# for compara*ility stu#ies
A /ften technically challenging for routine use
C1a$acte$i;ation te"t" *o$ Ma% $o!uct"
B +$ima$y St$uctu$e
A !C+MS :epti#e Maps
A N3terminal Se8uencing
A &erification of C3terminus
A Disulfi#e Bon# Determination
A ;lycan Map
A 'ntact Mass Determination
B Secon!a$y an! Te$tia$y St$uctu$e
A F$'R
A Far 9& CD
A Fluorescence
A Near 9& CD
B Ot1e$"
A Non3re#uce# CE3SDS
A CEX32:!C0 lo, p2
A DSC
A (9C
A SE3M(!S
A E1tinction Coefficient
A E1cipients
A :rocess impurities
B ,unctiona# c1a$acte$i;ation
A (ntigen *in#ing
A (##itional cell3*ase# assays
A Epitope mapping
A Fc^ R'0 R''' *in#ing
A (DCC
A CDC
A FcRn *in#ing
+$o!uction +o#yc#ona# anti%o!ie" Seen in !etai# un!e$ t1e toic o* antigen
$ea$ation Unit I
C1a$acte$i"ation o* +o#yc#ona# anti%o!ie"
!asis of polyclonality
Responses are polyclonal in nature as each clone some,hat speciali-es in pro#ucing anti*o#ies
against a gi.en epitope0 an# *ecause0 each antigen contains multiple epitopes0 each of ,hich in
turn can *e recogni-e# *y more than one clone of B cells. But0 to *e a*le to react to innumera*le
antigens0 as ,ell as0 multiple constituent epitopes0 the immune system re8uires the a*ility to
recogni-e a .ery great num*er of epitopes in all0 i.e.0 there shoul# *e a great #i.ersity of B cell
clones.
C#ona#ity o* B ce##"
Memory an# na_.e B cells normally e1ist in relati.ely small num*ers. (s the *o#y nee#s to *e
a*le to respon# to a large num*er of potential pathogens0 it maintains a pool of B cells ,ith a
,i#e range of specificities. Conse8uently0 ,hile there is almost al,ays at least one B nai.e or
memory" cell capa*le of respon#ing to any gi.en epitope of all that the immune system can
react against"0 there are .ery fe, e1act #uplicates. 2o,e.er0 ,hen a single B cell encounters an
antigen to ,hich it can *in#0 it can proliferate .ery rapi#ly. Such a group of cells ,ith i#entical
specificity to,ar#s the epitope is kno,n as a clone0 an# is #eri.e# from a common 4mother4
cell. (ll the 4#aughter4 B cells match the original 4mother4 cell in their epitope specificity0 an#
they secrete anti*o#ies ,ith i#entical paratopes. So0 in this conte1t0 a polyclonal response is one
in ,hich multiple clones of B cells react to the same antigen.
Sing#e antigen contain" mu#ti#e o'e$#aing eitoe"
Blin# Monks E1amining an Elephant: (n allegory for the polyclonal response: Each clone or
anti*o#y recogni-es #ifferent parts of a single0 larger antigen
( single antigen can *e thought of as a se8uence of multiple o.erlapping epitopes. Many uni8ue
B cell clones may *e a*le to *in# to the in#i.i#ual epitopes. $his imparts e.en greater
multiplicity to the o.erall response. (ll of these B cells can *ecome acti.ate# an# pro#uce large
colonies of plasma cell clones0 each of ,hich can secrete up to <BBB anti*o#y molecules against
each epitope per secon#.
Mu#ti#e c#one" $ecogni;e "ing#e eitoe
'n a##ition to #ifferent B cells reacting to different epitopes on the same antigen0 B cells
*elonging to #ifferent clones may also *e a*le to react to the same epitope. (n epitope that can
*e attacke# *y many #ifferent B cells is sai# to *e highly immunogenic. 'n these cases0 the
binding affinities for respecti.e epitope3paratope pairs .ary0 ,ith some B cell clones pro#ucing
anti*o#ies that *in# strongly to the epitope0 an# others pro#ucing anti*o#ies that *in# ,eakly.
C#ona# "e#ection
For more #etails on lymph no#es0 germinal centers of lymph no#es an# clonal selection of B
cells0 see !ymph no#e0 ;erminal center0 Clonal selection.
$he clones that *in# to a particular epitope ,ith greater strength are more likely to *e selected
for further proliferation in the germinal centers of the follicles in .arious lymphoi# tissues like
the lymph no#es. $his is not unlike natural selection: clones are selecte# for their fitness to
attack the epitopes strength of *in#ing" on the encountere# pathogen. Khat makes the analogy
e.en stronger is that the B lymphocytes ha.e to compete ,ith each other for signals that
promote their sur.i.al in the germinal centers.
4i'e$"ity o* B ce## c#one"
(lthough there are many #i.erse pathogens0 many of ,hich are constantly mutating0 it is a
surprise that a majority of in#i.i#uals remain free of infections. $hus0 maintenance of health
re8uires the *o#y to recogni-e all pathogens antigens they present or pro#uce" likely to e1ist.
$his is achie.e# *y maintaining a pool of immensely large a*out <B
>
" clones of B cells0 each of
,hich reacts against a specific epitope *y recogni-ing an# pro#ucing anti*o#ies against it.
2o,e.er0 at any gi.en time .ery fe, clones actually remain recepti.e to their specific epitope.
$hus0 appro1imately <B
?
#ifferent epitopes can *e recogni-e# *y all the B cell clones com*ine#.
Moreo.er0 in a lifetime0 an in#i.i#ual usually re8uires the generation of anti*o#ies against .ery
fe, antigens in comparison ,ith the num*er that the *o#y can recogni-e an# respon# against.
Significance of the phenomenon
Inc$ea"e! $o%a%i#ity o* $ecogni;ing any antigen
'f an antigen can *e recogni-e# *y more than one component of its structure0 it is less likely to
*e 4misse#4 *y the immune system. Mutation of pathogenic organisms can result in mo#ification
of antigen`an#0 hence0 epitope`structure. 'f the immune system 4remem*ers4 ,hat the other
epitopes look like0 the antigen0 an# the organism0 ,ill still *e recogni-e# an# su*jecte# to the
*o#yFs immune response. $hus0 the polyclonal response ,i#ens the range of pathogens that can
*e recogni-e#.
Limitation o* immune "y"tem again"t $ai!#y mutating 'i$u"e"
$he clone < that got stimulate# *y first antigen gets stimulate# *y secon# antigen0 too0 ,hich
*est *in#s ,ith nai.e cell of clone I. 2o,e.er0 anti*o#ies pro#uce# *y plasma cells of clone <
inhi*it the proliferation of clone I.
Many .iruses un#ergo fre8uent mutations that result in changes in amino aci# composition of
their important proteins. Epitopes locate# on the protein may also un#ergo alterations in the
process. Such an altere# epitope *in#s less strongly ,ith the anti*o#ies specific to the unaltere#
epitope that ,oul# ha.e stimulate# the immune system. $his is unfortunate *ecause somatic
hypermutation #oes gi.e rise to clones capa*le of pro#ucing solu*le anti*o#ies that ,oul# ha.e
*oun# the altere# epitope a.i#ly enough to neutrali-e it. But these clones ,oul# consist of nai.e
cells ,hich are not allo,e# to proliferate *y the ,eakly *in#ing anti*o#ies pro#uce# *y the
priorly stimulate# clone. $his #octrine is kno,n as the original antigenic sin. $his phenomenon
comes into play particularly in immune responses against influen-a0 #engue an# 2'& .iruses.
$his limitation0 ho,e.er0 is not impose# by the phenomenon of polyclonal response0 *ut rather0
against it *y an immune response that is *iase# in fa.or of e1perience# memory cells against the
4no.ice4 nai.e cells.
Inc$ea"e! c1ance" o* autoimmune $eaction"
'n autoimmunity the immune system ,rongly recogni-es certain nati.e molecules in the *o#y as
foreign self-antigen"0 an# mounts an immune response against them. Since these nati.e
molecules0 as normal parts of the *o#y0 ,ill naturally al,ays e1ist in the *o#y0 the attacks
against them can get stronger o.er time akin to secon#ary immune response". Moreo.er0 many
organisms e1hi*it molecular mimicry0 ,hich in.ol.es sho,ing those antigens on their surface
that are antigenically similar to the host proteins. $his has t,o possi*le conse8uences: first0
either the organism ,ill *e spare# as a self antigen5 or secon#ly0 that the anti*o#ies pro#uce#
against it ,ill also *in# to the mimicke# nati.e proteins. $he anti*o#ies ,ill attack the self3
antigens an# the tissues har*oring them *y acti.ating .arious mechanisms like the complement
acti.ation an# anti*o#y3#epen#ent cell3me#iate# cytoto1icity. 2ence0 ,i#er the range of
anti*o#y3specificities0 greater the chance that one or the other ,ill react against self3antigens
nati.e molecules of the *o#y".
6IE76I?7
4i**icu#ty in $o!ucing monoc#ona# anti%o!ie"
Monoclonal anti*o#ies are structurally i#entical immunoglo*ulin molecules ,ith i#entical
epitope3specificity all of them *in# ,ith the same epitope ,ith same affinity" as against their
polyclonal counterparts ,hich ha.e .arying affinities for the same epitope. $hey are usually not
pro#uce# in a natural immune response0 *ut only in #isease# states like multiple myeloma0 or
through speciali-e# la*oratory techni8ues. Because of their specificity0 monoclonal anti*o#ies
are use# in certain applications to 8uantify or #etect the presence of su*stances ,hich act as
antigen for the monoclonal anti*o#ies"0 an# for targeting in#i.i#ual cells e.g. cancer cells".
Monoclonal anti*o#ies fin# use in .arious #iagnostic mo#alities see: ,estern *lot an#
immunofluorescence" an# therapies`particularly of cancer an# #iseases ,ith autoimmune
component. But0 since .irtually all responses in nature are polyclonal0 it makes pro#uction of
immensely useful monoclonal anti*o#ies less straightfor,ar#.
S4S6+AGE
:icture of an SDS3:(;E. $he molecular marker is in the left lane
S4S6+AGE0 "o!ium !o!ecy# "u#*ate o#yac$y#ami!e ge# e#ect$o1o$e"i"0 is a techni8ue
,i#ely use# in *iochemistry0 forensics0 genetics an# molecular *iology to separate proteins
accor#ing to their electrophoretic mo*ility a function of length of polypepti#e chain or
molecular ,eight". SDS gel electrophoresis of samples ha.e i#entical charge per unit mass #ue
to *in#ing of SDS results in fractionation *y si-e.
Procedure
Ti""ue $ea$ation
Samples may *e taken from ,hole tissue or from cell culture. 'n most cases0 soli# tissues are
first *roken #o,n mechanically using a *len#er for larger sample .olumes"0 using a
homogeni-er smaller .olumes"0 or *y sonicator. Cells may also *e *roken open *y one of the
a*o.e mechanical metho#s. 2o,e.er0 it shoul# *e note# that *acteria0 .irus or en.ironmental
samples can *e the source of protein an# thus Kestern *lotting is not restricte# to cellular stu#ies
only.
( com*ination of *iochemical an# mechanical techni8ues A inclu#ing .arious types of filtration
an# centrifugation A can *e use# to separate #ifferent cell compartments an# organelles.
$he solution of proteins to *e analy-e# is mi1e# ,ith SDS0 an anionic #etergent ,hich #enatures
secon#ary an# nonA#isulfi#eAlinke# tertiary structures0 an# applies a negati.e charge to each
protein in proportion to its mass. 2eating the samples to at least EB #egrees C shakes up the
molecules0 helping SDS to *in#.
( tracking #ye may *e a##e# to the protein solution of a si-e smaller than protein" to allo, the
e1perimenter to track the progress of the protein solution through the gel #uring the
electrophoretic run.
+$ea$ing ac$y#ami!e ge#"
$he gels generally consist of acrylami#e0 *isacrylami#e0 SDS0 an# a $ris3Cl *uffer ,ith a#juste#
p2. $he solution is #egasse# un#er a .acuum to pre.ent air *u**les #uring polymeri-ation.
(mmonium persulfate an# $EMED are a##e# ,hen the gel is rea#y to *e polymeri-e#. $he
separating or resol.ing gel is usually more *asic an# has a higher polyacrylami#e content than
the loa#ing gel.
;els are polymeri-e# in a gel caster. First the separating gel is poure# an# allo,e# to
polymeri-e. Ne1t a thin layer of isopropanol is a##e#. Ne1t the loa#ing gel is poure# an# a com*
is place# to create the ,ells. (fter the loa#ing gel is polymeri-e# the com* can *e remo.e# an#
the gel is rea#y for electrophoresis.
E#ect$o1o$e"i"
First the ano#e an# catho#e *uffers are prepare#. $he ano#e *uffer usually contains $ris3Cl0
#istille# #eioni-e# ,ater an# is a#juste# to a higher p2 than the catho#e *uffer. $he catho#e
*uffer contains SDS0 $ris0 $ricine0 an# #istille# #eioni-e# ,ater.
6?7

6=7
$he electrophoresis apparatus is set up ,ith catho#e *uffer co.ering the gel in the negati.e
electro#e cham*er0 an# ano#e *uffer in the lo,er positi.e electro#e cham*er. Ne1t0 the
#enature# sample proteins are a##e# to the ,ells one en# of the gel ,ith a syringe or pipette.
Finally0 the apparatus is hooke# up to a po,er source un#er appropriate running con#itions to
separate the protein *an#s.
(n electric fiel# is applie# across the gel0 causing the negati.ely3charge# proteins to migrate
across the gel to,ar#s the positi.e G" electro#e ano#e". Depen#ing on their si-e0 each protein
,ill mo.e #ifferently through the gel matri1: short proteins ,ill more easily fit through the pores
in the gel0 ,hile larger ones ,ill ha.e more #ifficulty they encounter more resistance". (fter a
set amount of time usually a fe, hours3 though this #epen#s on the .oltage applie# across the
gel5 higher .oltages run faster *ut ten# to pro#uce some,hat poorer resolution"0 the proteins ,ill
ha.e #ifferentially migrate# *ase# on their si-e5 smaller proteins ,ill ha.e tra.ele# farther #o,n
the gel0 ,hile larger ones ,ill ha.e remaine# closer to the point of origin. $herefore0 proteins
may *e separate# roughly accor#ing to si-e an# therefore0 molecular ,eight"0 certain
glycoproteins *eha.e anomalously on SDS gels.
Staining
$,o SDS3:(;E3gels after a complete# run
Follo,ing electrophoresis0 the gel may *e staine# most commonly ,ith Coomassie Brilliant
Blue R3ILB or sil.er stain"0 allo,ing .isuali-ation of the separate# proteins0 or processe# further
e.g. Kestern *lot". (fter staining0 #ifferent proteins ,ill appear as #istinct *an#s ,ithin the gel.
't is common to run molecular ,eight si-e markers of kno,n molecular ,eight in a separate
lane in the gel0 in or#er to cali*rate the gel an# #etermine the ,eight of unkno,n proteins *y
comparing the #istance tra.ele# relati.e to the marker. $he gel is actually forme# *ecause the
acrylami#e solution contains a small amount0 generally a*out < part in %L of *isacrylami#e0
,hich can form cross3links *et,een t,o polyacrylami#e molecules. $he ratio of acrylami#e to
*isacrylami#e can *e .arie# for special purposes. $he acrylami#e concentration of the gel can
also *e .arie#0 generally in the range from LZ to ILZ. !o,er percentage gels are *etter for
resol.ing .ery high molecular ,eight proteins0 ,hile much higher percentages are nee#e# to
resol.e smaller proteins. Determining ho, much of the .arious solutions to mi1 together to
make gels of particular acrylami#e concentration can *e #one on line
;el electrophoresis is usually the first choice as an assay of protein purity #ue to its relia*ility
an# ease. $he presence of SDS an# the #enaturing step causes proteins to *e separate# solely
*ase# on si-e. False negati.es an# positi.es are possi*le. ( comigrating contaminant can appear
as the same *an# as the #esire# protein. $his comigration coul# also cause a protein to run at a
#ifferent position or to not *e a*le to penetrate the gel. $his is ,hy it is important to stain the
entire gel inclu#ing the stacking section. Coomassie Brilliant Blue ,ill also *in# ,ith less
affinity to glycoproteins an# fi*rous proteins0 ,hich interferes ,ith 8uantification.
Chemical ingredients and their roles
Polyacrylamide gel (PAG) ha# *een kno,n as a potential em*e##ing me#ium for sectioning
tissues as early as <>E@. $,o in#epen#ent groups0 Da.is an# Raymon#0 employe# :(; in
electrophoresis in <>L>. 't possesses se.eral electrophoretically #esira*le features that make it a
.ersatile me#ium. :(;E separates protein molecules accor#ing to *oth si-e an# charge. 't is a
synthetic gel0 thermo3sta*le0 transparent0 strong0 relati.ely chemically inert0 can *e prepare#
,ith a ,i#e range of a.erage pore si-es. $he pore si-e of a gel is #etermine# *y t,o factors0 the
total amount of acrylami#e present Z$" $ C $otal acrylami#e3*isacrylami#e monomer
concentration" an# the amount of cross3linker ZC" C C Crosslinker concentration". :ore si-e
#ecreases ,ith increasing Z$5 ,ith cross3linking0 LZC gi.es the smallest pore si-e. (ny
increase or #ecrease in ZC increases the pore si-e0 as pore si-e ,ith respect to ZC is a para*olic
function ,ith .erte1 as LZC. $his appears to *e *ecause of nonhomogeneous *un#ling of
stran#s in the gel.
$his gel material can also ,ithstan# high .oltage gra#ients0 feasi*le for .arious staining an#
#estaining proce#ures0 an# can *e #igeste# to e1tract separate# fractions or #rie# for
autora#iography an# permanent recor#ing. D'SC electrophoresis utili-es gels of #ifferent pore
si-es. $he name D'SC ,as #eri.e# from the #iscontinuities in the electrophoretic matri1 an#
coinci#entally from the #iscoi# shape of the separate# -ones of ions. $here are t,o layers of gel0
namely stacking or spacer gel0 an# resol.ing or separating gel.
Stac5ing ge#
$he stacking gel is a large pore :(; @Z$". $his gel is prepare# ,ith $ris+2Cl *uffer p2 E.= of
a*out I p2 units lo,er than that of electrophoresis *uffer $ris+;lycine". $hese con#itions
pro.i#e an en.ironment for Nohlrausch reactions #etermining molar con#ucti.ity0 as a result0
SDS3coate# proteins are concentrate# to se.eral fol# an# a thin starting -one of the or#er of
<> am is achie.e# in a fe, minutes. $his gel is cast o.er the resol.ing gel. $he height of the
stacking gel region is al,ays maintaine# more than #ou*le the height an# the .olume of the
sample to *e applie#. $his is *ase# on isotachophoresis.
C1emica# ing$e!ient"
T$i" (t$i" (1y!$o&y met1y#) aminomet1ane) (C
4
H
??
NO
@
A m92 ?2?3?4)3 't has *een
use# as a *uffer *ecause it is an innocuous su*stance to most proteins. 'ts pNa is =.% at
IB bC0 making it a .ery satisfactory *uffer in the p2 range from roughly ? to >.
G#ycine (Amino Acetic Aci!) (C
2
H
B
NO
2
A m92 CB30C)3 ;lycine has *een use# as the
source of trailing ion or slo, ion *ecause its pNa is >.E> an# mo*ility of glycinate are
such that the effecti.e mo*ility can *e set at a .alue *elo, that of the slo,est kno,n
proteins of net negati.e charge in the p2 range. $he minimum p2 of this range is
appro1imately =.B.
Ac$y#ami!e (C
@
H
B
NOA m92 C?30D)3 't is a ,hite crystalline po,#er. Khile #issol.ing in
,ater0 autopolymeri-ation of acrylami#e takes place. 't is a slo, spontaneous process *y
,hich acrylami#e molecules join together *y hea# on tail fashion. But in presence of free
ra#icals generating system0 acrylami#e monomers are acti.ate# into a free3ra#ical state.
$hese acti.ate# monomers polymerise 8uickly an# form long chain polymers. $his kin#
of reaction is kno,n as &inyl a##ition polymerisation. ( solution of these polymer
chains *ecomes .iscous *ut #oes not form a gel0 *ecause the chains simply sli#e o.er
one another. ;el formation re8uires hooking .arious chains together. (crylami#e is a
neuroto1in. 't is also essential to store acrylami#e in a cool #ark an# #ry place to re#uce
autopolymerisation an# hy#rolysis.
Bi"ac$y#ami!e (NEN=6Met1y#ene%i"ac$y#ami!e) (C
C
H
?0
N
2
O
2
A m92 ?B43?C)3
Bisacrylami#e is the most fre8uently use# cross linking agent for poly acrylami#e gels.
Chemically it is thought of ha.ing t,o3acrylami#e molecules couple# hea# to hea# at
their non3reacti.e en#s.
So!ium 4o!ecy# Su#*ate (S4S) (C
?2
H
2B
NaO
4
SA m92 2DD3@D)3 SDS is the most common
#issociating agent use# to #enature nati.e proteins to in#i.i#ual polypepti#es. Khen a
protein mi1ture is heate# to <BB bC in presence of SDS0 the #etergent ,raps aroun# the
polypepti#e *ack*one. 't *in#s to polypepti#es in a constant ,eight ratio of <.@ g+g of
polypepti#e. 'n this process0 the intrinsic charges of polypepti#es *ecomes negligi*le
,hen compare# to the negati.e charges contri*ute# *y SDS. $hus polypepti#es after
treatment *ecomes a ro# like structure possessing a uniform charge #ensity0 that is same
net negati.e charge per unit length. Mo*ilities of these proteins ,ill *e a linear function
of the logarithms of their molecular ,eights.
Kithout SDS0 #ifferent proteins ,ith similar molecular ,eights ,oul# migrate
#ifferently #ue to #ifferences in mass charge ratio0 as each protein has an isoelectric point
an# molecular ,eight particular to its primary structure. $his is kno,n as Nati.e :(;E.
(##ing SDS sol.es this pro*lem0 as it *in#s to an# unfol#s the protein0 gi.ing a near
uniform negati.e charge along the length of the polypepti#e.
Ammonium e$"u#*ate (A+S) N
I
2
=
S
I
/
=
5 mK: II=.I". (:S is an initiator for gel
formation.
TEME4 (NE NE N=E N=6tet$amet1y#et1y#ene!iamine" C
E
2
<E
N
I
5 mK: <<E.I<".
Chemical polymerisation of acrylami#e gel is use# for SDS3:(;E. 't can *e initiate# *y
ammonium persulfate an# the 8uaternary amine0 N0N0NF0NF3tetramethylethylene#iamine
$EMED". $he rate of polymerisation an# the properties of the resulting gel #epen#s on
the concentration of (:S an# $EMED. 'ncreasing the amount of (:S an# $EMED
results in a #ecrease in the a.erage polymer chain length0 an increase in gel tur*i#ity an#
a #ecrease in gel elasticity. Decreasing the amount of initiators sho,s the re.erse effect.
$he lo,est catalytic concentrations that ,ill allo, polymerisation in the optimal perio#
of time shoul# *e use#. (:S an# $EMED are use#0 appro1imately in e8uimolar
concentrations in the range of < to <B mM.
C1emica#" *o$ $oce""ing an! 'i"ua#i;ation
$he follo,ing chemicals are use# for processing of the gel an# the protein samples .isuali-e# in
it:
B$omo1eno# %#ue (B+B" %F0%40LF0L4 tetra*romophenolsulfonphthalein" (C
?F
H
?0
B$
4
O
B
SA
m92 66F3FF). B:B is the uni.ersal marker #ye. :roteins an# nucleic aci#s are mostly
colourless. Khen they are su*jecte# to electrophoresis0 it is important to stop the run
*efore they run off the gel. B:B is the most commonly employe# tracking #ye0 *ecause
it is .ia*le in alkali an# neutral p20 it is a small molecule0 it is ionisa*le an# it is
negati.ely charge# a*o.e p2 @.E an# hence mo.es to,ar#s the ano#e. Being a small
molecule it mo.es ahea# of most proteins an# nucleic aci#s. (s it reaches the ano#ic en#
of the electrophoresis me#ium electrophoresis is stoppe#. 't can *in# ,ith proteins
,eakly an# gi.e *lue colour.
G#yce$o# (C
@
H
D
O
@
A m92 F230F)3 't is a preser.ati.e an# a ,eighing agent. (##ition of
glycerol IB3%B or LBZ" is often recommen#e# for the storage of en-ymes. ;lycerol
maintains the protein solution at .ery lo, temperature0 ,ithout free-ing. 't also helps to
,eigh #o,n the sample into the ,ells ,ithout *eing sprea# ,hile loa#ing.
Cooma""ie B$i##iant B#ue -62B0 (CBB)(C
4B
H
44
N
@
NaO
C
S
2
A m92 D2B3FC). CBB is the
most popular protein stain. 't is an anionic #ye0 ,hich *in#s ,ith proteins non3
specifically. $he structure of CBB is pre#ominantly non3polar. So is usually use#
B.BILZ" in methanolic solution @BZ" an# acetic aci# ?Z". :roteins in the gel are fi1e#
*y acetic aci# an# simultaneously staine#. $he e1cess #ye incorporate# in the gel can *e
remo.e# *y #estaining ,ith the same solution *ut ,ithout the #ye. $he proteins are
#etecte# as *lue *an#s on a clear *ackgroun#. (s SDS is also anionic0 it may interfere
,ith staining process. $herefore0 large .olume of staining solution is recommen#e#0
appro1imately ten times the .olume of the gel.
n6Butano# (C
4
H
?0
OA m92 C43?2)3 Kater saturate# *utanol is use# as an o.erlay solution
on the resol.ing gel.
4it1iot1$eito# (4TTA C
4
H
?0
O
2
S
2
A m92 ?B432B)3 D$$ is a re#ucing agent use# to #isrupt
#isulfi#e *on#s to ensure the protein is fully #enature# *efore loa#ing on the gel5
ensuring the protein runs uniformly. $ra#itionally the to1ic an# less potent I3
mercaptoethanol ,as use#.
Reducing SS"PA#$
Besi#es the a##ition of SDS0 proteins may optionally *e *riefly heate# to near *oiling in the
presence of a re#ucing agent0 such as #ithiothreitol D$$" or tra#itionally I3mercaptoethanol
*eta3mercaptoethanol+BME"0 ,hich further #enatures the proteins *y re#ucing #isulfi#e
linkages0 thus o.ercoming some forms of tertiary protein fol#ing0 an# *reaking up 8uaternary
protein structure oligomeric su*units". $his is kno,n as re#ucing SDS3:(;E0 an# is most
commonly use#. Non3re#ucing SDS3:(;E no *oiling an# no re#ucing agent" may *e use#
,hen nati.e structure is important in further analysis e.g. en-yme acti.ity0 sho,n *y the use of
-ymograms". For e1ample0 .uantitati.e reparati.e nati.e continuous olyacrylami#e gel
electrophoresis R:NC3:(;E" is a ne, metho# for separating nati.e metalloproteins in
comple1 *iological matrices.
Silver staining
Si#'e$ "taine! S4S +o#yac$y#ami!e ge#"
'n the <@th century the sil.er staining techni8ue ,as #e.elope# for colouring the surface of
glass. 't has *een use# e1tensi.ely for this purpose since the <Eth century. $he colour pro#uce#
*y the early sil.er stains range# *et,een light yello, an# an orange3re#. Camillo ;olgi
perfecte# the sil.er staining for the stu#y of the ner.ous system. ;olgiFs metho# stains a limite#
num*er of cells at ran#om in their entirety.
6<@7
$he e1act chemical mechanism *y ,hich this
happens is still largely unkno,n.
6<L7
Sil.er staining ,as intro#uce# *y Nerenyi an# ;allyas as a
sensiti.e proce#ure to #etect trace amounts of proteins in gels.
6<E7
$he techni8ue has *een
e1ten#e# to the stu#y of other *iological macromolecules that ha.e *een separate# in a .ariety
of supports.
6<?7
Classical Coomassie Brilliant Blue staining can usually #etect a LB ng protein
*an#0 Sil.er staining increases the sensiti.ity typically LB times. Many .aria*les can influence
the colour intensity an# e.ery protein has its o,n staining characteristics5 clean glass,are0 pure
reagents an# ,ater of highest purity are the key points to successful staining.
6<=7
!uffer systems
Most protein separations are performe# using a 4#iscontinuous4 *uffer system that significantly
enhances the sharpness of the *an#s ,ithin the gel. During electrophoresis in a #iscontinuous gel
system0 an ion gra#ient is forme# in the early stage of electrophoresis that causes all of the
proteins to focus into a single sharp *an#. $his occurs in a region of the gel that has larger pores
so that the gel matri1 #oes not retar# the migration #uring the focusing or 4stacking4 e.ent.
Negati.e ions from the *uffer in the tank then 4outrun4 the SDS3co.ere# protein 4stack4 an#
eliminate the ion gra#ient so that the proteins su*se8uently separate *y the sie.ing action in the
lo,er0 4resol.ing4 region of the gel.
Many people continue to use a tris3glycine or 4!aemmli4 *uffering system that stacks at a p2 of
E.= an# resol.es at a p2 of c=.%3>.B. $hese p2s promote #isulfi#e *on# formation *et,een
cysteine resi#ues in the proteins0 especially ,hen they are present at high concentrations *ecause
the pNa of cysteine ranges from =3> an# *ecause re#ucing agent present in the loa#ing *uffer
#oesnFt co3migrate ,ith the proteins. Recent a#.ances in *uffering technology alle.iate this
pro*lem *y resol.ing the proteins at a p2 ,ell *elo, the pNa of cysteine e.g.0 *is3tris0 p2 E.L"
an# inclu#e re#ucing agents e.g. so#ium *isulfite" that mo.e into the gel ahea# of the proteins
to maintain a re#ucing en.ironment. (n a##itional *enefit of using *uffers ,ith lo,er p2s is
that the acrylami#e gel is more sta*le so the gels can *e store# for long perio#s of time *efore
use.
6<>76IB7
SS gradient gel electrophoresis of proteins
Migration of proteins in SDS gels of .arying acrylami#e concentrations Z$". $he migration of
nine proteins ranging from >@ kDa to <@.@ kDa is sho,n. Stacking an# unstacking occurs
continuously in the gel0 for e.ery protein at a #ifferent gel concentration. $he #otte# line
in#icates the #iscontinuity at the ;lyd+Cld mo.ing *oun#ary. :roteins *et,een the fast lea#ing
electrolyte an# the slo, trailing electrolyte are not #ilute# *y #iffusion.
(s .oltage is applie#0 the anions an# negati.ely charge# sample molecules" migrate to,ar# the
positi.e electro#e ano#e" in the lo,er cham*er0 the lea#ing ion is Cld high mo*ility an# high
concentration"5 glycinate is the trailing ion lo, mo*ility an# lo, concentration". SDS3protein
particles #o not migrate freely at the *or#er *et,een the Cld of the gel *uffer an# the ;lyd of the
catho#e *uffer. Frie#rich Nohlrausch foun# that /hmFs la, also applies to #issol.e# electrolytes.
Because of the .oltage #rop *et,een the Cl3 an# ;lycine3*uffers0 proteins are compresse#
stacke#" into micrometer thin layers. $he *oun#ary mo.es through a pore gra#ient an# the
protein stack gra#ually #isperses #ue to an frictional resistance increase of the gel matri1.
Stacking an# unstacking occurs continuously in the gra#ient gel0 for e.ery protein at a #ifferent
position. For a complete protein unstacking the polyacrylami#e3gel concentration must e1cee#
<EZ $. $he t,o3gel system of 4!aemmli4 is a simple gra#ient gel. $he p2 #iscontinuity of the
*uffers is of no significance for the separation 8uality0 an# a 4stacking3gel4 ,ith a #ifferent p2 is
not nee#e#.
9e"te$n %#ot
Kestern *lot analysis of proteins separate# *y SDS3:(;E.
$he 9e"te$n %#ot alternati.ely0 $otein immuno%#ot" is an e1tremely useful analytical
techni8ue use# to #etect specific proteins in a gi.en sample of tissue homogenate or e1tract. 't
uses gel electrophoresis to separate nati.e or #enature# proteins *y the length of the polypepti#e
#enaturing con#itions" or *y the %3D structure of the protein nati.e+ non3#enaturing
con#itions". $he proteins are then transferre# to a mem*rane typically nitrocellulose or :&DF"0
,here they are pro*e# #etecte#" using anti*o#ies specific to the target protein.
$here are no, many reagent companies that speciali-e in pro.i#ing anti*o#ies *oth monoclonal
an# polyclonal anti*o#ies" against tens of thousan#s of #ifferent proteins. Commercial
anti*o#ies can *e e1pensi.e0 although the un*oun# anti*o#y can *e reuse# *et,een
e1periments. $his metho# is use# in the fiel#s of molecular *iology0 *iochemistry0
immunogenetics an# other molecular *iology #isciplines.
/ther relate# techni8ues inclu#e using anti*o#ies to #etect proteins in tissues an# cells *y
immunostaining an# en-yme3linke# immunosor*ent assay E!'S(".
$he metho# originate# from the la*oratory of ;eorge Stark at Stanfor#. $he name Western blot
,as gi.en to the techni8ue *y K. Neal Burnette an# is a play on the name Southern *lot0 a
techni8ue for DN( #etection #e.elope# earlier *y E#,in Southern. Detection of RN( is terme#
northern *lotting an# the #etection of post3translational mo#ification of protein is terme# eastern
*lotting.
Steps in a %estern &lot
Ti""ue $ea$ation
Samples may *e taken from ,hole tissue or from cell culture. 'n most cases0 soli# tissues are
first *roken #o,n mechanically using a *len#er for larger sample .olumes"0 using a
homogeni-er smaller .olumes"0 or *y sonication. Cells may also *e *roken open *y one of the
a*o.e mechanical metho#s. 2o,e.er0 it shoul# *e note# that *acteria0 .irus or en.ironmental
samples can *e the source of protein an# thus Kestern *lotting is not restricte# to cellular stu#ies
only.
(ssorte# #etergents0 salts0 an# *uffers may *e employe# to encourage lysis of cells an# to
solu*ili-e proteins. :rotease an# phosphatase inhi*itors are often a##e# to pre.ent the #igestion
of the sample *y its o,n en-ymes. $issue preparation is often #one at col# temperatures to a.oi#
protein #enaturing an# #egra#ation.
( com*ination of *iochemical an# mechanical techni8ues A inclu#ing .arious types of filtration
an# centrifugation A can *e use# to separate #ifferent cell compartments an# organelles.
Ge# e#ect$o1o$e"i"
$he proteins of the sample are separate# using gel electrophoresis. Separation of proteins may
*e *y isoelectric point p'"0 molecular ,eight0 electric charge0 or a com*ination of these factors.
$he nature of the separation #epen#s on the treatment of the sample an# the nature of the gel.
$his is a .ery useful ,ay to #etermine a protein.
By far the most common type of gel electrophoresis employs polyacrylami#e gels an# *uffers
loa#e# ,ith so#ium #o#ecyl sulfate SDS". SDS3:(;E SDS polyacrylami#e gel
electrophoresis" maintains polypepti#es in a #enature# state once they ha.e *een treate# ,ith
strong re#ucing agents to remo.e secon#ary an# tertiary structure e.g. #isulfi#e *on#s 6S3S7 to
sulfhy#ryl groups 6S2 an# S27" an# thus allo,s separation of proteins *y their molecular
,eight. Sample# proteins *ecome co.ere# in the negati.ely charge# SDS an# mo.e to the
positi.ely charge# electro#e through the acrylami#e mesh of the gel. Smaller proteins migrate
faster through this mesh an# the proteins are thus separate# accor#ing to si-e usually measure#
in kilo#altons0 kDa". $he concentration of acrylami#e #etermines the resolution of the gel 3 the
greater the acrylami#e concentration the *etter the resolution of lo,er molecular ,eight
proteins. $he lo,er the acrylami#e concentration the *etter the resolution of higher molecular
,eight proteins. :roteins tra.el only in one #imension along the gel for most *lots.
Samples are loa#e# into ells in the gel. /ne lane is usually reser.e# for a marker or ladder0 a
commercially a.aila*le mi1ture of proteins ha.ing #efine# molecular ,eights0 typically staine#
so as to form .isi*le0 coloure# *an#s. Khen .oltage is applie# along the gel0 proteins migrate
into it at #ifferent spee#s. $hese #ifferent rates of a#.ancement #ifferent electrophoretic
mobilities" separate into bands ,ithin each lane.
't is also possi*le to use a t,o3#imensional I3D" gel ,hich sprea#s the proteins from a single
sample out in t,o #imensions. :roteins are separate# accor#ing to isoelectric point p2 at ,hich
they ha.e neutral net charge" in the first #imension0 an# accor#ing to their molecular ,eight in
the secon# #imension.
T$an"*e$
'n or#er to make the proteins accessi*le to anti*o#y #etection0 they are mo.e# from ,ithin the
gel onto a mem*rane ma#e of nitrocellulose or poly!inylidene difluoride "P#$F". $he
mem*rane is place# on top of the gel0 an# a stack of filter papers place# on top of that. $he
entire stack is place# in a *uffer solution ,hich mo.es up the paper *y capillary action0 *ringing
the proteins ,ith it. (nother metho# for transferring the proteins is calle# electro*lotting an#
uses an electric current to pull proteins from the gel into the :&DF or nitrocellulose mem*rane.
$he protein mo.e from ,ithin the gel onto the mem*rane ,hile maintaining the organi-ation
they ha# ,ithin the gel. (s a result of this 4*lotting4 process0 the proteins are e1pose# on a thin
surface layer for #etection see *elo,". Both .arieties of mem*rane are chosen for their non3
specific protein *in#ing properties i.e. *in#s all proteins e8ually ,ell". :rotein *in#ing is *ase#
upon hy#ropho*ic interactions0 as ,ell as charge# interactions *et,een the mem*rane an#
protein. Nitrocellulose mem*ranes are cheaper than :&DF0 *ut are far more fragile an# #o not
stan# up ,ell to repeate# pro*ings.
$he uniformity an# o.erall effecti.eness of transfer of protein from the gel to the mem*rane can
*e checke# *y staining the mem*rane ,ith Coomassie Brilliant Blue or :onceau S #yes.
:onceau S is the more common of the t,o0 #ue to :onceau SFs higher sensiti.ity an# its ,ater
solu*ility makes it easier to su*se8uently #estain an# pro*e the mem*rane as #escri*e# *elo,.
B#oc5ing
Since the mem*rane has *een chosen for its a*ility to *in# protein an# as *oth anti*o#ies an# the
target are proteins0 steps must *e taken to pre.ent interactions *et,een the mem*rane an# the
anti*o#y use# for #etection of the target protein. Blocking of non3specific *in#ing is achie.e# *y
placing the mem*rane in a #ilute solution of protein 3 typically %3LZ Bo.ine serum al*umin
BS(" or non3fat #ry milk *oth are ine1pensi.e" in $ris3Buffere# Saline $BS"0 ,ith a minute
percentage of #etergent such as $,een IB or $riton X3<BB. $he protein in the #ilute solution
attaches to the mem*rane in all places ,here the target proteins ha.e not attache#. $hus0 ,hen
the anti*o#y is a##e#0 there is no room on the mem*rane for it to attach other than on the
*in#ing sites of the specific target protein. $his re#uces 4noise4 in the final pro#uct of the
Kestern *lot0 lea#ing to clearer results0 an# eliminates false positi.es.
4etection
During the #etection process the mem*rane is 4pro*e#4 for the protein of interest ,ith a
mo#ifie# anti*o#y ,hich is linke# to a reporter en-yme0 ,hich ,hen e1pose# to an appropriate
su*strate #ri.es a colourimetric reaction an# pro#uces a colour. For a .ariety of reasons0 this
tra#itionally takes place in a t,o3step process0 although there are no, one3step #etection
metho#s a.aila*le for certain applications.
T:o "te"
:rimary anti*o#y
(nti*o#ies are generate# ,hen a host species or immune cell culture is e1pose# to the protein of
interest or a part thereof". Normally0 this is part of the immune response0 ,hereas here they are
har.este# an# use# as sensiti.e an# specific #etection tools that *in# the protein #irectly.
(fter *locking0 a #ilute solution of primary anti*o#y generally *et,een B.L an# L
micrograms+m!" is incu*ate# ,ith the mem*rane un#er gentle agitation. $ypically0 the solution
is compose# of *uffere# saline solution ,ith a small percentage of #etergent0 an# sometimes
,ith po,#ere# milk or BS(. $he anti*o#y solution an# the mem*rane can *e seale# an#
incu*ate# together for any,here from %B minutes to o.ernight. 't can also *e incu*ate# at
#ifferent temperatures0 ,ith ,armer temperatures *eing associate# ,ith more *in#ing0 *oth
specific to the target protein0 the 4signal4" an# non3specific 4noise4".
Secon#ary anti*o#y
(fter rinsing the mem*rane to remo.e un*oun# primary anti*o#y0 the mem*rane is e1pose# to
another anti*o#y0 #irecte# at a species3specific portion of the primary anti*o#y. (nti*o#ies come
from animal sources or animal source# hy*ri#oma cultures"5 an anti3mouse secon#ary ,ill *in#
to almost any mouse3source# primary anti*o#y0 ,hich allo,s some cost sa.ings *y allo,ing an
entire la* to share a single source of mass3pro#uce# anti*o#y0 an# pro.i#es far more consistent
results. $his is kno,n as a secon#ary anti*o#y0 an# #ue to its targeting properties0 ten#s to *e
referre# to as 4anti3mouse04 4anti3goat04 etc. $he secon#ary anti*o#y is usually linke# to *iotin
or to a reporter en-yme such as alkaline phosphatase or horsera#ish pero1i#ase. $his means that
se.eral secon#ary anti*o#ies ,ill *in# to one primary anti*o#y an# enhance the signal.
Most commonly0 a horsera#ish pero1i#ase3linke# secon#ary is use# to clea.e a
chemiluminescent agent0 an# the reaction pro#uct pro#uces luminescence in proportion to the
amount of protein. ( sensiti.e sheet of photographic film is place# against the mem*rane0 an#
e1posure to the light from the reaction creates an image of the anti*o#ies *oun# to the *lot. (
cheaper *ut less sensiti.e approach utili-es a @3chloronaphthol stain ,ith <Z hy#rogen
pero1i#e5 reaction of pero1i#e ra#icals ,ith @3chloronaphthol pro#uces a #ark *ro,n stain that
can *e photographe# ,ithout using speciali-e# photographic film.
(s ,ith the E!'S:/$ an# E!'S( proce#ures0 the en-yme can *e pro.i#e# ,ith a su*strate
molecule that ,ill *e con.erte# *y the en-yme to a colore# reaction pro#uct that ,ill *e .isi*le
on the mem*rane see the figure *elo, ,ith *lue *an#s".
(nother metho# of secon#ary anti*o#y #etection utili-es a near3infrare# N'R" fluorophore3
linke# anti*o#y. !ight pro#uce# from the e1citation of a fluorescent #ye is static0 making
fluorescent #etection a more precise an# accurate measure of the #ifference in signal pro#uce#
*y la*ele# anti*o#ies *oun# to proteins on a Kestern *lot. :roteins can *e accurately 8uantifie#
*ecause the signal generate# *y the #ifferent amounts of proteins on the mem*ranes is measure#
in a static state0 as compare# to chemiluminescence0 in ,hich light is measure# in a #ynamic
state.
( thir# alternati.e is to use a ra#ioacti.e la*el rather than an en-yme couple# to the secon#ary
anti*o#y0 such as la*eling an anti*o#y3*in#ing protein like %taphylococcus :rotein ( or
Strepta.i#in ,ith a ra#ioacti.e isotope of io#ine. Since other metho#s are safer0 8uicker0 an#
cheaper0 this metho# is no, rarely use#5 ho,e.er0 an a#.antage of this approach is the
sensiti.ity of auto3ra#iography *ase# imaging0 ,hich ena*les highly accurate protein
8uantification ,hen com*ine# ,ith optical soft,are e.g. /pti8uant".
One "te
2istorically0 the pro*ing process ,as performe# in t,o steps *ecause of the relati.e ease of
pro#ucing primary an# secon#ary anti*o#ies in separate processes. $his gi.es researchers an#
corporations huge a#.antages in terms of fle1i*ility0 an# a##s an amplification step to the
#etection process. ;i.en the a#.ent of high3throughput protein analysis an# lo,er limits of
#etection0 ho,e.er0 there has *een interest in #e.eloping one3step pro*ing systems that ,oul#
allo, the process to occur faster an# ,ith less consuma*les. $his re8uires a pro*e anti*o#y
,hich *oth recogni-es the protein of interest an# contains a #etecta*le la*el0 pro*es ,hich are
often a.aila*le for kno,n protein tags. $he primary pro*e is incu*ate# ,ith the mem*rane in a
manner similar to that for the primary anti*o#y in a t,o3step process0 an# then is rea#y for #irect
#etection after a series of ,ash steps.
Kestern *lot using ra#ioacti.e #etection system
Ana#y"i"
(fter the un*oun# pro*es are ,ashe# a,ay0 the Kestern *lot is rea#y for #etection of the pro*es
that are la*ele# an# *oun# to the protein of interest. 'n practical terms0 not all Kesterns re.eal
protein only at one *an# in a mem*rane. Si-e appro1imations are taken *y comparing the
staine# *an#s to that of the marker or la##er loa#e# #uring electrophoresis. $he process is
repeate# for a structural protein0 such as actin or tu*ulin0 that shoul# not change *et,een
samples. $he amount of target protein is in#e1e# to the structural protein to control *et,een
groups. $his practice ensures correction for the amount of total protein on the mem*rane in case
of errors or incomplete transfers.
Co#o$imet$ic !etection
$he colorimetric #etection metho# #epen#s on incu*ation of the Kestern *lot ,ith a su*strate
that reacts ,ith the reporter en-yme such as pero1i#ase" that is *oun# to the secon#ary
anti*o#y. $his con.erts the solu*le #ye into an insolu*le form of a #ifferent color that
precipitates ne1t to the en-yme an# there*y stains the mem*rane. De.elopment of the *lot is
then stoppe# *y ,ashing a,ay the solu*le #ye. :rotein le.els are e.aluate# through
#ensitometry ho, intense the stain is" or spectrophotometry.
C1emi#umine"cent !etection
Chemiluminescent #etection metho#s #epen# on incu*ation of the Kestern *lot ,ith a su*strate
that ,ill luminesce ,hen e1pose# to the reporter on the secon#ary anti*o#y. $he light is then
#etecte# *y photographic film0 an# more recently *y CCD cameras ,hich capture a #igital
image of the Kestern *lot. $he image is analyse# *y #ensitometry0 ,hich e.aluates the relati.e
amount of protein staining an# 8uantifies the results in terms of optical #ensity. Ne,er soft,are
allo,s further #ata analysis such as molecular ,eight analysis if appropriate stan#ar#s are use#.
-a!ioacti'e !etection
Ra#ioacti.e la*els #o not re8uire en-yme su*strates0 *ut rather allo, the placement of me#ical
X3ray film #irectly against the Kestern *lot ,hich #e.elops as it is e1pose# to the la*el an#
creates #ark regions ,hich correspon# to the protein *an#s of interest see image to the right".
$he importance of ra#ioacti.e #etections metho#s is #eclining
0
*ecause it is .ery e1pensi.e0
health an# safety risks are high0 an# EC! enhance# chemiluminescence" pro.i#es a useful
alternati.e.
,#uo$e"cent !etection
$he fluorescently la*ele# pro*e is e1cite# *y light an# the emission of the e1citation is then
#etecte# *y a photosensor such as CCD camera e8uippe# ,ith appropriate emission filters ,hich
captures a #igital image of the Kestern *lot an# allo,s further #ata analysis such as molecular
,eight analysis an# a 8uantitati.e Kestern *lot analysis. Fluorescence is consi#ere# to *e among
the most sensiti.e #etection metho#s for *lotting analysis.
Secon!a$y $o%ing
/ne major #ifference *et,een nitrocellulose an# :&DF mem*ranes relates to the a*ility of each
to support 4stripping4 anti*o#ies off an# reusing the mem*rane for su*se8uent anti*o#y pro*es.
Khile there are ,ell3esta*lishe# protocols a.aila*le for stripping nitrocellulose mem*ranes0 the
stur#ier :&DF allo,s for easier stripping0 an# for more reuse *efore *ackgroun# noise limits
e1periments. (nother #ifference is that0 unlike nitrocellulose0 :&DF must *e soake# in >LZ
ethanol0 isopropanol or methanol *efore use. :&DF mem*ranes also ten# to *e thicker an# more
resistant to #amage #uring use.
'" gel electrophoresis
I3#imensional SDS3:(;E uses the principles an# techni8ues outline# a*o.e. I3D SDS3:(;E0
as the name suggests0 in.ol.es the migration of polypepti#es in I #imensions. For e1ample0 in
the first #imension polypepti#es are separate# accor#ing to isoelectric point0 ,hile in the secon#
#imension polypepti#es are separate# accor#ing to their molecular ,eight. $he isoelectric point
of a gi.en protein is #etermine# *y the relati.e num*er of positi.ely e.g. lysine an# arginine"
an# negati.ely e.g. glutamate an# aspartate" charge# amino aci#s0 ,ith negati.ely charge#
amino aci#s contri*uting to a high isoelectric point an# positi.ely charge# amino aci#s
contri*uting to a lo, isoelectric point. Samples coul# also *e separate# first un#er nonre#ucing
con#itions using SDS3:(;E an# un#er re#ucing con#itions in the secon# #imension0 ,hich
*reaks apart #isulfi#e *on#s that hol# su*units together. SDS3:(;E might also *e couple# ,ith
urea3:(;E for a I3#imensional gel.
'n principle0 this metho# allo,s for the separation of all cellular proteins on a single large gel. (
major a#.antage of this metho# is that it often #istinguishes *et,een #ifferent isoforms of a
particular protein 3 e.g. a protein that has *een phosphorylate# *y a##ition of a negati.ely
charge# group". :roteins that ha.e *een separate# can *e cut out of the gel an# then analyse# *y
mass spectrometry0 ,hich i#entifies the protein.
:lease refer to reference articles for e1amples of the application of I3D SDS :(;E.
(edical diagnostic applications
$he confirmatory 2'& test employs a Kestern *lot to #etect anti32'& anti*o#y in a
human serum sample. :roteins from kno,n 2'&3infecte# cells are separate# an# *lotte#
on a mem*rane as a*o.e. $hen0 the serum to *e teste# is applie# in the primary anti*o#y
incu*ation step5 free anti*o#y is ,ashe# a,ay0 an# a secon#ary anti3human anti*o#y
linke# to an en-yme signal is a##e#. $he staine# *an#s then in#icate the proteins to
,hich the patientFs serum contains anti*o#y.
( Kestern *lot is also use# as the #efiniti.e test for Bo.ine spongiform encephalopathy
BSE0 commonly referre# to as Fma# co, #iseaseF".
Some forms of !yme #isease testing employ Kestern *lotting.
Kestern *lot can also *e use# as a confirmatory test for 2epatitis B infection.
'n .eterinary me#icine0 Kestern *lot is sometimes use# to confirm F'&G status in cats.
Immunoe#ect$o1o$e"i"
Immunoe#ect$o1o$e"i" is a general name for a num*er of *iochemical metho#s for separation
an# characteri-ation of proteins *ase# on electrophoresis an# reaction ,ith anti*o#ies. (ll
.ariants of immunoelectrophoresis re8uire immunoglo*ulins0 also kno,n as anti*o#ies reacting
,ith the proteins to *e separate# or characteri-e#. $he metho#s ,ere #e.elope# an# use#
e1tensi.ely #uring the secon# half of the IBth century. 'n some,hat chronological or#er:
'mmunoelectrophoretic analysis one3#imensional immunoelectrophoresis ad modum ;ra*ar"0
crosse# immunoelectrophoresis t,o3#imensional 8uantitati.e immunoelectrophoresis ad
modum Clarke an# Freeman or ad modum !aurell"0 rocket3immunoelectrophoresis one3
#imensional 8uantitati.e immunoelectrophoresis ad modum !aurell"0 fuse# rocket
immunoelectrophoresis ad modum S.en#sen an# 2ar*oe0 affinity immunoelectrophoresis ad
modum Beg32ansen.
(garose as < Z gel sla*s of a*out < mm thickness *uffere# at high p2 aroun# =.E" is
tra#itionally preferre# for the electrophoresis as ,ell as the reaction ,ith anti*o#ies. $he agarose
,as chosen as the gel matri1 *ecause it has large pores allo,ing free passage an# separation of
proteins0 *ut pro.i#es an anchor for the immunoprecipitates of protein an# specific anti*o#ies.
$he high p2 ,as chosen *ecause anti*o#ies are practically immo*ile at high p2.
'mmunoprecipitates may *e seen in the ,et agarose gel0 *ut are staine# ,ith protein stains like
Coomassie Brilliant Blue in the #rie# gel. 'n contrast to SDS3gel electrophoresis0 the
electrophoresis in agarose allo,s nati.e con#itions0 preser.ing the nati.e structure an# acti.ities
of the proteins un#er in.estigation0 therefore immunoelectrophoresis allo,s characteri-ation of
en-yme acti.ities an# ligan# *in#ing etc in a##ition to electrophoretic separation.
$he immunoe#ect$o1o$etic ana#y"i" ad modum G$a%a$ is the classical metho# of
immunoelectrophoresis. :roteins are separate# *y electrophoresis0 then anti*o#ies are applie# in
a trough ne1t to the separate# proteins an# immunoprecipitates are forme# after a perio# of
#iffusion of the separate# proteins an# anti*o#ies against each other. $he intro#uction of the
immunoelectrophoretic analysis ga.e a great *oost to protein chemistry0 some of the .ery first
results ,ere the resolution of proteins in *iological flui#s an# *iological e1tracts. (mong the
important o*ser.ations ma#e ,ere the great num*er of #ifferent proteins in serum0 the e1istence
of se.eral immunoglo*ulin classes an# their electrophoretic heterogeneity.
C$o""e! immunoe#ect$o1o$e"i" is also calle# t,o3#imensional 8uantitati.e
immunoelectrophoresis ad modum Clarke an# Freeman or ad modum !aurell. 'n this metho# the
proteins are first separate# #uring the first #imension electrophoresis0 then instea# of the
#iffusion to,ar#s the anti*o#ies0 the proteins are electrophorese# into an anti*o#y3containing
gel in the secon# #imension. 'mmunoprecipitation ,ill take place #uring the secon# #imension
electrophorsis an# the immunoprecipitates ha.e a characteristic *ell3shape0 each precipitate
representing one antigen0 the position of the precipitate *eing #epen#ent on the amount of
protein as ,ell as the amount of specific anti*o#y in the gel0 so relati.e 8uantification can *e
performe#. $he sensiti.ity an# resol.ing po,er of crosse# immunoelectrophoresis is than that of
the classical immunoelectrophoretic analysis an# there are multiple .ariations of the techni8ue
useful for .arious purposes. Crosse# immunoelectrophoresis has *een use# for stu#ies of
proteins in *iological flui#s0 particularly human serum0 an# *iological e1tracts.
-oc5et immunoe#ect$o1o$e"i" is one3#imensional 8uantitati.e immunoelectrophoresis. $he
metho#s has *een use# for 8uantitation of human serum proteins *efore automate# metho#s
*ecame a.aila*le.
,u"e! $oc5et immunoe#ect$o1o$e"i" is a mo#ification of one3#imensional 8uantitati.e
immunoelectrophorsis use# for #etaile# measurement of proteins in fractions from protein
separation e1periments.
A**inity immunoe#ect$o1o$e"i" is *ase# on changes in the electrophoretic pattern of proteins
through *iospecific interaction or comple1 formation ,ith other macromolecules or ligan#s.
(ffinity immunoelectrophoresis has *een use# for estimation of *in#ing constants0 as for
instance ,ith lectins or for characteri-ation of proteins ,ith specific features like glycan content
or ligan# *in#ing. Some .ariants of affinity immunoelectrophoresis are similar to affinity
chromatography *y use of immo*ili-e# ligan#s.
$he open structure of the immunoprecipitate in the agarose gel ,ill allo, a##itional *in#ing of
ra#ioacti.ely la*ele# anti*o#ies to re.eal specific proteins. $his .ariation has *een use# for
i#entification of allergens through reaction ,ith 'gE.
$,o factors #etermine that immunoelectrophoretic metho#s are not ,i#ely use#. First they are
rather ,ork intensi.e an# re8uire some manual e1pertise. Secon# they re8uire rather large
amounts of polyclonal anti*o#ies. $o#ay gel electrophoresis follo,e# *y electro*lotting is the
preferre# metho# for protein characteri-ation *ecause its ease of operation0 its high sensiti.ity0
an# its lo, re8uirement for specific anti*o#ies. 'n a##ition proteins are separate# *y gel
electrophoresis on the *asis of their apparent molecular ,eight0 ,hich is not accomplishe# *y
immunoelectrophoresis0 *ut ne.ertheless immunoelectrophoretic metho#s are still useful ,hen
non3re#ucing con#itions are nee#e#.
+$otein u$i*ication
+$otein u$i*ication is a series of processes inten#e# to isolate a single type of protein from a
comple1 mi1ture. :rotein purification is .ital for the characterisation of the function0 structure
an# interactions of the protein of interest. $he starting material is usually a *iological tissue or a
micro*ial culture. $he .arious steps in the purification process may free the protein from a
matri1 that confines it0 separate the protein an# non3protein parts of the mi1ture0 an# finally
separate the #esire# protein from all other proteins. Separation of one protein from all others is
typically the most la*orious aspect of protein purification. Separation steps e1ploit #ifferences in
protein si-e0 physico3chemical properties an# *in#ing affinity.
Purpose
:urification may *e preparati!e or analytical. :reparati.e purifications aim to pro#uce a
relati.ely large 8uantity of purifie# proteins for su*se8uent use. E1amples inclu#e the
preparation of commercial pro#ucts such as en-ymes e.g. lactase"0 nutritional proteins e.g. soy
protein isolate"0 an# certain *iopharmaceuticals e.g. insulin". (nalytical purification pro#uces a
relati.ely small amount of a protein for a .ariety of research or analytical purposes0 inclu#ing
i#entification0 8uantification0 an# stu#ies of the proteinFs structure0 post3translational
mo#ifications an# function. (mong the first purifie# proteins ,ere urease an# Concana.alin (.
Strategies
Choice of a starting material is key to the #esign of a purification process. 'n a plant or animal0 a
particular protein usually isnFt #istri*ute# homogeneously throughout the *o#y5 #ifferent organs
or tissues ha.e higher or lo,er concentrations of the protein. 9se of only the tissues or organs
,ith the highest concentration #ecreases the .olumes nee#e# to pro#uce a gi.en amount of
purifie# protein. 'f the protein is present in lo, a*un#ance0 or if it has a high .alue0 scientists
may use recom*inant DN( technology to #e.elop cells that ,ill pro#uce large 8uantities of the
#esire# protein this is kno,n as an e1pression system". Recom*inant e1pression allo,s the
protein to *e tagge#0 e.g. *y a 2is3tag0 to facilitate purification0 ,hich means that the
purification can *e #one in fe,er steps. 'n a##ition0 recom*inant e1pression usually starts ,ith a
higher fraction of the #esire# protein than is present in a natural source.
(n analytical purification generally utili-es three properties to separate proteins. First0 proteins
may *e purifie# accor#ing to their isoelectric points *y running them through a p2 gra#e# gel or
an ion e1change column. Secon#0 proteins can *e separate# accor#ing to their si-e or molecular
,eight .ia si-e e1clusion chromatography or *y SDS3:(;E so#ium #o#ecyl sulfate3
polyacrylami#e gel electrophoresis" analysis. :roteins are often purifie# *y using ID3:(;E an#
are then analyse# *y pepti#e mass fingerprinting to esta*lish the protein i#entity. $his is .ery
useful for scientific purposes an# the #etection limits for protein are no,a#ays .ery lo, an#
nanogram amounts of protein are sufficient for their analysis. $hir#ly0 proteins may *e separate#
*y polarity+hy#ropho*icity .ia high performance li8ui# chromatography or re.erse#3phase
chromatography.
$valuating purification yield
$he most general metho# to monitor the purification process is *y running a SDS3:(;E of the
#ifferent steps. $his metho# only gi.es a rough measure of the amounts of #ifferent proteins in
the mi1ture0 an# it is not a*le to #istinguish *et,een proteins ,ith similar apparent molecular
,eight.
'f the protein has a #istinguishing spectroscopic feature or an en-ymatic acti.ity0 this property
can *e use# to #etect an# 8uantify the specific protein0 an# thus to select the fractions of the
separation0 that contains the protein. 'f anti*o#ies against the protein are a.aila*le then ,estern
*lotting an# E!'S( can specifically #etect an# 8uantify the amount of #esire# protein. Some
proteins function as receptors an# can *e #etecte# #uring purification steps *y a ligan# *in#ing
assay0 often using a ra#ioacti.e ligan#.
'n or#er to e.aluate the process of multistep purification0 the amount of the specific protein has
to *e compare# to the amount of total protein. $he latter can *e #etermine# *y the Bra#for# total
protein assay or *y a*sor*ance of light at I=B nm0 ho,e.er some reagents use# #uring the
purification process may interfere ,ith the 8uantification. For e1ample0 imi#a-ole commonly
use# for purification of polyhisti#ine3tagge# recom*inant proteins" is an amino aci# analogue
an# at lo, concentrations ,ill interfere ,ith the *icinchoninic aci# BC(" assay for total protein
8uantification. 'mpurities in lo,3gra#e imi#a-ole ,ill also a*sor* at I=B nm0 resulting in an
inaccurate rea#ing of protein concentration from 9& a*sor*ance.
(nother metho# to *e consi#ere# is Surface :lasmon Resonance S:R". S:R can #etect *in#ing
of la*el free molecules on the surface of a chip. 'f the #esire# protein is an anti*o#y0 *in#ing can
*e translate# to #irectly to the acti.ity of the protein. /ne can e1press the acti.e concentration of
the protein as the percent of the total protein. S:R can *e a po,erful metho# for 8uickly
#etermining protein acti.ity an# o.erall yiel#. 't is a po,erful technology that re8uires an
instrument to perform.
(ethods of protein purification
$he metho#s use# in protein purification can roughly *e #i.i#e# into analytical an# preparati.e
metho#s. $he #istinction is not e1act0 *ut the #eci#ing factor is the amount of protein that can
practically *e purifie# ,ith that metho#. (nalytical metho#s aim to #etect an# i#entify a protein
in a mi1ture0 ,hereas preparati.e metho#s aim to pro#uce large 8uantities of the protein for
other purposes0 such as structural *iology or in#ustrial use. 'n general0 the preparati.e metho#s
can *e use# in analytical applications0 *ut not the other ,ay aroun#.
)* $+traction
Depen#ing on the source0 the protein has to *e *rought into solution *y *reaking the tissue or
cells containing it. $here are se.eral metho#s to achie.e this: Repeate# free-ing an# tha,ing0
sonication0 homogeni-ation *y high pressure0 filtration either .ia cellulose3*ase# #epth filters or
cross3flo, filtration
6<7
"0 or permea*ili-ation *y organic sol.ents. $he metho# of choice #epen#s
on ho, fragile the protein is an# ho, stur#y the cells are. (fter this e1traction process solu*le
proteins ,ill *e in the sol.ent0 an# can *e separate# from cell mem*ranes0 DN( etc. *y
centrifugation. $he e1traction process also e1tracts proteases0 ,hich ,ill start #igesting the
proteins in the solution. 'f the protein is sensiti.e to proteolysis0 it is usually #esira*le to procee#
8uickly0 an# keep the e1tract coole#0 to slo, #o,n proteolysis.
'* Precipitation and differential solu&ili,ation
'n *ulk protein purification0 a common first step to isolate proteins is precipitation ,ith
ammonium sulfate N2
@
"
I
S/
@
. $his is performe# *y a##ing increasing amounts of ammonium
sulfate an# collecting the #ifferent fractions of precipitate protein. /ne a#.antage of this metho#
is that it can *e performe# ine1pensi.ely ,ith .ery large .olumes.
$he first proteins to *e purifie# are ,ater3solu*le proteins. :urification of integral mem*rane
proteins re8uires #isruption of the cell mem*rane in or#er to isolate any one particular protein
from others that are in the same mem*rane compartment. Sometimes a particular mem*rane
fraction can *e isolate# first0 such as isolating mitochon#ria from cells *efore purifying a protein
locate# in a mitochon#rial mem*rane. ( #etergent such as so#ium #o#ecyl sulfate SDS" can *e
use# to #issol.e cell mem*ranes an# keep mem*rane proteins in solution #uring purification5
ho,e.er0 *ecause SDS causes #enaturation0 mil#er #etergents such as $riton X3<BB or C2(:S
can *e use# to retain the proteinFs nati.e conformation #uring complete purification.
-* .ltracentrifugation
Cent$i*ugation is a process that uses centrifugal force to separate mi1tures of particles of
.arying masses or #ensities suspen#e# in a li8ui#. Khen a .essel typically a tu*e or *ottle"
containing a mi1ture of proteins or other particulate matter0 such as *acterial cells0 is rotate# at
high spee#s0 the angular momentum yiel#s an out,ar# force to each particle that is proportional
to its mass. $he ten#ency of a gi.en particle to mo.e through the li8ui# *ecause of this force is
offset *y the resistance the li8ui# e1erts on the particle. $he net effect of 4spinning4 the sample
in a centrifuge is that massi.e0 small0 an# #ense particles mo.e out,ar# faster than less massi.e
particles or particles ,ith more 4#rag4 in the li8ui#. Khen suspensions of particles are 4spun4 in
a centrifuge0 a 4pellet4 may form at the *ottom of the .essel that is enriche# for the most
massi.e particles ,ith lo, #rag in the li8ui#. Non3compacte# particles still remaining mostly in
the li8ui# are calle# the 4supernatant4 an# can *e remo.e# from the .essel to separate the
supernatant from the pellet. $he rate of centrifugation is specifie# *y the angular acceleration
applie# to the sample0 typically measure# in comparison to the g. 'f samples are centrifuge# long
enough0 the particles in the .essel ,ill reach e8uili*rium ,herein the particles accumulate
specifically at a point in the .essel ,here their *uoyant #ensity is *alance# ,ith centrifugal
force. Such an 4e8uili*rium4 centrifugation can allo, e1tensi.e purification of a gi.en particle.
Sucrose gra#ient centrifugation ` a linear concentration gra#ient of sugar typically sucrose0
glycerol0 or a silica *ase# #ensity gra#ient me#ia0 like :ercoll" is generate# in a tu*e such that
the highest concentration is on the *ottom an# lo,est on top. :ercoll is a tra#emark o,ne# *y
;E 2ealthcare companies. ( protein sample is then layere# on top of the gra#ient an# spun at
high spee#s in an ultracentrifuge. $his causes hea.y macromolecules to migrate to,ar#s the
*ottom of the tu*e faster than lighter material. During centrifugation in the a*sence of sucrose0
as particles mo.e farther an# farther from the center of rotation0 they e1perience more an# more
centrifugal force the further they mo.e0 the faster they mo.e". $he pro*lem ,ith this is that the
useful separation range of ,ithin the .essel is restricte# to a small o*ser.a*le ,in#o,. Spinning
a sample t,ice as long #oesnFt mean the particle of interest ,ill go t,ice as far0 in fact0 it ,ill go
significantly further. 2o,e.er0 ,hen the proteins are mo.ing through a sucrose gra#ient0 they
encounter li8ui# of increasing #ensity an# .iscosity. ( properly #esigne# sucrose gra#ient ,ill
counteract the increasing centrifugal force so the particles mo.e in close proportion to the time
they ha.e *een in the centrifugal fiel#. Samples separate# *y these gra#ients are referre# to as
4rate -onal4 centrifugations. (fter separating the protein+particles0 the gra#ient is then
fractionate# an# collecte#.
/* Chromatographic methods
9sually a protein purification protocol contains one or more chromatographic steps. $he *asic
proce#ure in chromatography is to flo, the solution containing the protein through a column
packe# ,ith .arious materials. Different proteins interact #ifferently ,ith the column material0
an# can thus *e separate# *y the time re8uire# to pass the column0 or the con#itions re8uire# to
elute the protein from the column. 9sually proteins are #etecte# as they are coming off the
column *y their a*sor*ance at I=B nm. Many #ifferent chromatographic metho#s e1ist:
a3 Si;e e&c#u"ion c1$omatog$a1y
Ge# e$meation c1$omatog$a1y
Chromatography can *e use# to separate protein in solution or #enaturing con#itions *y using
porous gels. $his techni8ue is kno,n as si-e e1clusion chromatography. $he principle is that
smaller molecules ha.e to tra.erse a larger .olume in a porous matri1. Conse8uentially0 proteins
of a certain range in si-e ,ill re8uire a .aria*le .olume of eluent sol.ent" *efore *eing
collecte# at the other en# of the column of gel.
'n the conte1t of protein purification0 the eluant is usually poole# in #ifferent test tu*es. (ll test
tu*es containing no measura*le trace of the protein to purify are #iscar#e#. $he remaining
solution is thus ma#e of the protein to purify an# any other similarly3si-e# proteins.
%3 Sea$ation %a"e! on c1a$ge o$ 1y!$o1o%icity
2y#ropho*ic 'nteraction Chromatography
c3 Ion e&c1ange c1$omatog$a1y
'on e1change chromatography separates compoun#s accor#ing to the nature an# #egree of their
ionic charge. $he column to *e use# is selecte# accor#ing to its type an# strength of charge.
(nion e1change resins ha.e a positi.e charge an# are use# to retain an# separate negati.ely
charge# compoun#s0 ,hile cation e1change resins ha.e a negati.e charge an# are use# to
separate positi.ely charge# molecules.
Before the separation *egins a *uffer is pumpe# through the column to e8uili*rate the opposing
charge# ions. 9pon injection of the sample0 solute molecules ,ill e1change ,ith the *uffer ions
as each competes for the *in#ing sites on the resin. $he length of retention for each solute
#epen#s upon the strength of its charge. $he most ,eakly charge# compoun#s ,ill elute first0
follo,e# *y those ,ith successi.ely stronger charges. Becauses of the nature of the separating
mechanism0 p20 *uffer type0 *uffer concentration0 an# temperature all play important roles in
controlling the separation.
'on e1change chromatography is a .ery po,erful tool for use in protein purification an# is
fre8uently use# in *oth analytical an# preparati.e separations.
Nickel3affinity column. $he resin is *lue since it has *oun# nickel.
!3 A**inity c1$omatog$a1y
(ffinity Chromatography is a separation techni8ue *ase# upon molecular conformation0 ,hich
fre8uently utili-es application specific resins. $hese resins ha.e ligan#s attache# to their
surfaces ,hich are specific for the compoun#s to *e separate#. Most fre8uently0 these ligan#s
function in a fashion similar to that of anti*o#y3antigen interactions. $his 4lock an# key4 fit
*et,een the ligan# an# its target compoun# makes it highly specific0 fre8uently generating a
single peak0 ,hile all else in the sample is unretaine#.
Many mem*rane proteins are glycoproteins an# can *e purifie# *y lectin affinity
chromatography. Detergent3solu*ili-e# proteins can *e allo,e# to *in# to a chromatography
resin that has *een mo#ifie# to ha.e a co.alently attache# lectin. :roteins that #o not *in# to the
lectin are ,ashe# a,ay an# then specifically *oun# glycoproteins can *e elute# *y a##ing a high
concentration of a sugar that competes ,ith the *oun# glycoproteins at the lectin *in#ing site.
Some lectins ha.e high affinity *in#ing to oligosacchari#es of glycoproteins that is har# to
compete ,ith sugars0 an# *oun# glycoproteins nee# to *e release# *y #enaturing the lectin.
e3 Meta# %in!ing
( common techni8ue in.ol.es engineering a se8uence of E to = histi#ines into the N3 or
C3terminal of the protein. $he polyhisti#ine *in#s strongly to #i.alent metal ions such as nickel
an# co*alt. $he protein can *e passe# through a column containing immo*ili-e# nickel ions0
,hich *in#s the polyhisti#ine tag. (ll untagge# proteins pass through the column. $he protein
can *e elute# ,ith imi#a-ole0 ,hich competes ,ith the polyhisti#ine tag for *in#ing to the
column0 or *y a #ecrease in p2 typically to @.L"0 ,hich #ecreases the affinity of the tag for the
resin. Khile this proce#ure is generally use# for the purification of recom*inant proteins ,ith an
engineere# affinity tag such as a E12is tag or ClontechFs 2($ tag"0 it can also *e use# for
natural proteins ,ith an inherent affinity for #i.alent cations.
*3 Immunoa**inity c1$omatog$a1y
'mmunoaffinity chromatography uses the specific *in#ing of an anti*o#y to the target protein to
selecti.ely purify the protein. $he proce#ure in.ol.es immo*ili-ing an anti*o#y to a column
material0 ,hich then selecti.ely *in#s the protein0 ,hile e.erything else flo,s through. $he
protein can *e elute# *y changing the p2 or the salinity. Because this metho# #oes not in.ol.e
engineering in a tag0 it can *e use# for proteins from natural sources.
B3 +u$i*ication o* a tagge! $otein
(##ing a tag to the protein such as RuB:S gi.es the protein a *in#ing affinity it ,oul# not
other,ise ha.e. 9sually the recom*inant protein is the only protein in the mi1ture ,ith this
affinity0 ,hich ai#s in separation. $he most common tag is the 2isti#ine3tag 2is3tag"0 that has
affinity to,ar#s nickel or co*alt ions. $hus *y immo*ili-ing nickel or co*alt ions on a resin0 an
affinity support that specifically *in#s to histi#ine3tagge# proteins can *e create#. Since the
protein is the only component ,ith a 2is3tag0 all other proteins ,ill pass through the column0
an# lea.e the 2is3tagge# protein *oun# to the resin. $he protein is release# from the column in a
process calle# elution0 ,hich in this case in.ol.es a##ing imi#a-ole0 to compete ,ith the 2is3
tags for nickel *in#ing0 as it has a ring structure similar to histi#ine. $he protein of interest is
no, the major protein component in the elute# mi1ture0 an# can easily *e separate# from any
minor un,ante# contaminants *y a secon# step of purification0 such as si-e e1clusion
chromatography or R:32:!C.
(nother ,ay to tag proteins is to engineer an antigen pepti#e tag onto the protein0 an# then
purify the protein on a column or *y incu*ating ,ith a loose resin that is coate# ,ith an
immo*ili-e# anti*o#y. $his particular proce#ure is kno,n as immunoprecipitation.
'mmunoprecipitation is 8uite capa*le of generating an e1tremely specific interaction ,hich
usually results in *in#ing only the #esire# protein. $he purifie# tagge# proteins can then easily
*e separate# from the other proteins in solution an# later elute# *ack into clean solution.
Khen the tags are not nee#e# anymore0 they can *e clea.e# off *y a protease. $his often
in.ol.es engineering a protease clea.age site *et,een the tag an# the protein.
Concentration of the purified protein
( selecti.ely permea*le mem*rane can *e mounte# in a centrifuge tu*e. $he *uffer is force#
through the mem*rane *y centrifugation0 lea.ing the protein in the upper cham*er.
(t the en# of a protein purification0 the protein often has to *e concentrate#. Different metho#s
e1ist.
Lyo1i#i;ation
'f the solution #oesnFt contain any other solu*le component than the protein in 8uestion the
protein can *e lyophili-e# #rie#". $his is commonly #one after an 2:!C run. $his simply
remo.es all .olatile component lea.ing the proteins *ehin#.
U#t$a*i#t$ation
9ltrafiltration concentrates a protein solution using selecti.e permea*le mem*ranes. $he
function of the mem*rane is to let the ,ater an# small molecules pass through ,hile retaining
the protein. $he solution is force# against the mem*rane *y mechanical pump or gas pressure or
centrifugation.
Analytical
4enatu$ing6Con!ition E#ect$o1o$e"i"
;el electrophoresis is a common la*oratory techni8ue that can *e use# *oth as preparati.e an#
analytical metho#. $he principle of electrophoresis relies on the mo.ement of a charge# ion in
an electric fiel#. 'n practice0 the proteins are #enature# in a solution containing a #etergent
SDS". 'n these con#itions0 the proteins are unfol#e# an# coate# ,ith negati.ely charge#
#etergent molecules. $he proteins in SDS3:(;E are separate# on the sole *asis of their si-e.
'n analytical metho#s0 the protein migrate as *an#s *ase# on si-e. Each *an# can *e #etecte#
using stains such as Coomassie *lue #ye or sil.er stain. :reparati.e metho#s to purify large
amounts of protein0 re8uire the e1traction of the protein from the electrophoretic gel. $his
e1traction may in.ol.e e1cision of the gel containing a *an#0 or eluting the *an# #irectly off the
gel as it runs off the en# of the gel.
'n the conte1t of a purification strategy0 #enaturing con#ition electrophoresis pro.i#es an
impro.e# resolution o.er si-e e1clusion chromatography0 *ut #oes not scale to large 8uantity of
proteins in a sample as ,ell as the late chromatography columns.
Non64enatu$ing6Con!ition E#ect$o1o$e"i"
(n important non3#enaturing electrophoretic proce#ure for isolating *ioacti.e metalloproteins in
comple1 protein mi1tures is terme# F8uantitati.e nati.e continuous polyacrylami#e gel
electrophoresis R:NC3:(;E".
Met1o! *o$ concent$ation an! u$i*ication o* antigen" an! anti%o!ie"
'n carrying out a purification an#+or concentration step of antigens or anti*o#ies0 a su*strate is
immerse# in a first a8ueous me#ium containing a specifically reacting antigen to coat sai#
su*strate ,ith a monomolecular layer of sai# specifically reacting antigen. $he resulting coate#
su*strate is then immerse# in a secon# a8ueous me#ium containing immunologically reacti.e
anti*o#y specific to the antigen in the first a8ueous me#ium to comple1 sai# immunologically
reacti.e anti*o#y ,ith sai# specifically reacting antigen. $he resulting su*strate is then
immerse# in a reagent capa*le of clea.ing the immunological *on# *et,een sai#
immunologically reacti.e anti*o#y an# sai# specifically reacti.e antigen an# forming a solution
of sai# immunologically reacti.e anti*o#y in the immunological *on#3clea.ing solution an#
lea.ing sai# specifically reacting antigen coate# on sai# su*strate. $he metho# can *e re.erse#
for preparing a purifie# concentration of an immunologically reacti.e antigen ,here*y a
specifically reacting anti*o#y is su*stitute# for the antigen an# the correspon#ing
immunologically reacti.e antigen is su*stitute# for the anti*o#y.
Synt1e"i" o* Antigen" T1$oug1 C#one! Gene" 3 2un#re#s of genes in eukaryotes ha.e *een
clone# either from genomic DN( or from cDN(. $hese clone# genes inclu#e# a num*er of
genes for specific antigens0 an# in some cases ha.e *een use# for the synthesis of antigens
lea#ing to the preparation of .accines.
Follo,ing t,o e1amples can *e use# to illustrate the use of clone# genes for .accine preparation
i" C#oning o* Heatiti" B 'i$u" (HBY) genome. $he 2B& genome has *een clone# in the
plasmi# pBR%II an# propagate# in E. coli. From this clone0 antigen coul# *e pro#uce# in goo#
8uantity0 ,hich reacte# ,ith hepatitis B core anti*o#y 2B(*". $his has therefore *een use# to
pro#uce hepatitis B .accine0 ,hich ,as later appro.e# for mass .accination in se.eral countries.
(ii) C#oning o* 1uman ma#a$ia# gene. Despite the great menace an# threat to human health #ue
to malarial parasite :lasmo#ium falciparum0 no anti3malaria .accine0 coul# *e #e.elope# so far.
Recently ,ith cloning of a gene co#ing for surface protein of the sporo-oite of :. falciparum0
there is a hope for #e.eloping a .accine.
'n human host0 malarial parasite passes. through se.eral antigenically #istinct phases0 namely5
(i) "o$o;oite: the form in ,hich the parasite is injecte# ,ith mos8uito *ite5 sporo-oites enter
the li.er an# multiply an# #e.elop into
(ii) me$o;oite"0 ,hich in turn in.a#e an# multiply in re# *loo# cells5 small fraction of these
mero-oites in re# *loo# cells form
(iii) gametocyte"0 ,hich may *e picke# up *y a mos8uito to start another cycle.
Recent a#.ances in the research of artificial antigen ha.e sho,n that artificial antigens can
*e .alua*le approach for the treatment of some #iseases as ,ell as the #etection of pestici#e
resi#ues.By #irectly+in#irectly coupling hapten to an appropriate# carrier macromolecule"0
artificial antigen can in#uce animals to pro#uce hapten3specific anti*o#y. Base# on this
principle0 .arious .accines ha.e *een #e.elope#. More impotently0 ne, analytical metho#0
immunological analysis has also *een esta*lishe#. Comparing the con.entional technologies0
such as chromatographic metho#s0 this promising metho# offers an alternati.e ,ith high
specificity0 sensiti.ity0 simplicity an# suita*ility for the analysis of a large num*er of samples in
a short perio# of time.
ELISA
( >E3,ell microtiter plate *eing use# for E!'S(.
En;yme6#in5e! immuno"o$%ent a""ay ELISA"0 also kno,n as an en;yme immunoa""ay
EIA"0 is a *iochemical techni8ue use# mainly in immunology to #etect the presence of an
anti*o#y or an antigen in a sample. $he E!'S( has *een use# as a #iagnostic tool in me#icine
an# plant pathology0 as ,ell as a 8uality control check in .arious in#ustries. 'n simple terms0 in
E!'S(0 an unkno,n amount of antigen is affi1e# to a surface0 an# then a specific anti*o#y is
applie# o.er the surface so that it can *in# to the antigen. $his anti*o#y is linke# to an en-yme0
an# in the final step a su*stance is a##e# that the en-yme can con.ert to some #etecta*le signal0
most commonly a colour change in a chemical su*strate.
:erforming an E!'S( in.ol.es at least one anti*o#y ,ith specificity for a particular antigen. $he
sample ,ith an unkno,n amount of antigen is immo*ili-e# on a soli# support usually a
polystyrene microtiter plate" either non3specifically .ia a#sorption to the surface" or specifically
.ia capture *y another anti*o#y specific to the same antigen0 in a 4san#,ich4 E!'S(". (fter the
antigen is immo*ili-e# the #etection anti*o#y is a##e#0 forming a comple1 ,ith the antigen. $he
#etection anti*o#y can *e co.alently linke# to an en-yme0 or can itself *e #etecte# *y a
secon#ary anti*o#y ,hich is linke# to an en-yme through *ioconjugation. Bet,een each step the
plate is typically ,ashe# ,ith a mil# #etergent solution to remo.e any proteins or anti*o#ies that
are not specifically *oun#. (fter the final ,ash step the plate is #e.elope# *y a##ing an
en-ymatic su*strate to pro#uce a .isi*le signal0 ,hich in#icates the 8uantity of antigen in the
sample.
$ra#itional E!'S( typically in.ol.es chromogenic reporters an# su*strates ,hich pro#uce some
kin# of o*ser.a*le color change to in#icate the presence of antigen or analyte. Ne,er E!'S(3
like techni8ues utili-e fluorogenic0 electrochemiluminescent0 an# real3time :CR reporters to
create 8uantifia*le signals. $hese ne, reporters can ha.e .arious a#.antages inclu#ing higher
sensiti.ities an# multiple1ing. $echnically0 ne,er assays of this type are not strictly E!'S(s as
they are not 4en-yme3linke#4 *ut are instea# linke# to some non3en-ymatic reporter. 2o,e.er0
gi.en that the general principles in these assays are largely similar0 they are often groupe# in the
same category as E!'S(s.
Applications
E!'S( results using S3/'& ( neuramini#ase anti*o#y at < ag+ml to pro*e the immunogenic an#
the correspon#ing seasonal influen-a ( neuramini#ase pepti#es at LB0 <B0 I an# B ng+ml.
Because the E!'S( can *e performe# to e.aluate either the presence of antigen or the presence
of anti*o#y in a sample0 it is a useful tool for #etermining serum anti*o#y concentrations such
as ,ith the 2'& test
6%7
or Kest Nile &irus". 't has also foun# applications in the foo# in#ustry in
#etecting potential foo# allergens such as milk0 peanuts0 ,alnuts0 almon#s0 an# eggs. E!'S( can
also *e use# in to1icology as a rapi# presumpti.e screen for certain classes of #rugs.
$he E!'S( ,as the first screening test ,i#ely use# for 2'& *ecause of its high sensiti.ity. 'n an
E!'S(0 a personFs serum is #ilute# @BB3fol# an# applie# to a plate to ,hich 2'& antigens are
attache#. 'f anti*o#ies to 2'& are present in the serum0 they may *in# to these 2'& antigens.
$he plate is then ,ashe# to remo.e all other components of the serum. ( specially prepare#
4secon#ary anti*o#y4 ` an anti*o#y that *in#s to other anti*o#ies ` is then applie# to the
plate0 follo,e# *y another ,ash. $his secon#ary anti*o#y is chemically linke# in a#.ance to an
en-yme. $hus0 the plate ,ill contain en-yme in proportion to the amount of secon#ary anti*o#y
*oun# to the plate. ( su*strate for the en-yme is applie#0 an# catalysis *y the en-yme lea#s to a
change in color or fluorescence. E!'S( results are reporte# as a num*er5 the most contro.ersial
aspect of this test is #etermining the 4cut3off4 point *et,een a positi.e an# negati.e result.
( cut3off point may *e #etermine# *y comparing it ,ith a kno,n stan#ar#. 'f an E!'S( test is
use# for #rug screening at ,orkplace0 a cut3off concentration0 LB ng+m!0 for e1ample0 is
esta*lishe#0 an# a sample ,ill *e prepare# ,hich contains the stan#ar# concentration of analyte.
9nkno,ns that generate a signal that is stronger than the kno,n sample are 4positi.e4. $hose
that generate ,eaker signal are 4negati.e.4
History
Before the #e.elopment of the E!'S(0 the only option for con#ucting an immunoassay ,as
ra#ioimmunoassay0 a techni8ue using ra#ioacti.ely3la*ele# antigens or anti*o#ies. 'n
ra#ioimmunoassay0 the ra#ioacti.ity pro.i#es the signal ,hich in#icates ,hether a specific
antigen or anti*o#y is present in the sample. Ra#ioimmunoassay ,as first #escri*e# in a paper
*y Rosalyn Sussman Jalo, an# Solomon Berson pu*lishe# in <>EB.
Because ra#ioacti.ity poses a potential health threat0 a safer alternati.e ,as sought. ( suita*le
alternati.e to ra#ioimmunoassay ,oul# su*stitute a non3ra#ioacti.e signal in place of the
ra#ioacti.e signal. Khen en-ymes such as pero1i#ase" react ,ith appropriate su*strates such
as (B$S or %0%H0L0LH3$etramethyl*en-i#ine"0 this causes a change in color0 ,hich is use# as a
signal. 2o,e.er0 the signal has to *e associate# ,ith the presence of anti*o#y or antigen0 ,hich
is ,hy the en-yme has to *e linke# to an appropriate anti*o#y. $his linking process ,as
in#epen#ently #e.elope# *y Stratis (.rameas an# ;.B. :ierce. Since it is necessary to remo.e
any un*oun# anti*o#y or antigen *y ,ashing0 the anti*o#y or antigen has to *e fi1e# to the
surface of the container0 i.e. the immunosorbent has to *e prepare#. ( techni8ue to accomplish
this ,as pu*lishe# *y Ki#e an# Verker :orath in <>EE.
'n <>?<0 :eter :erlmann an# E.a Eng.all at Stockholm 9ni.ersity in S,e#en0 an# (nton
Schuurs an# Bauke .an Keemen in $he Netherlan#s0 in#epen#ently pu*lishe# papers ,hich
synthesi-e# this kno,le#ge into metho#s to perform E'(+E!'S(.
Types
GIn!i$ectG ELISA
$he steps of 4in#irect4 E!'S( follo,s the mechanism *elo,:3
( *uffere# solution of the antigen to *e teste# for is a##e# to each ,ell of a microtiter
plate0 ,here it is gi.en time to a#here to the plastic through charge interactions.
( solution of non3reacting protein0 such as *o.ine serum al*umin0 or casein is a##e# to
*lock any plastic surface in the ,ell that remains uncoate# *y the antigen.
Ne1t the primary anti*o#y0 generally in the form of serum is a##e#0 ,hich contains a
mi1ture of the serum #onorFs anti*o#ies0 of unkno,n concentration0 some of ,hich may
*in# specifically to the test antigen that is coating the ,ell.
(fter,ar#s0 a secon#ary anti*o#y is a##e#0 ,hich ,ill *in# any anti*o#y pro#uce# *y a
mem*er of the #onorFs species for e1ample0 an anti*o#y pro#uce# in a mouse that ,ill
*in# any ra**it anti*o#y". $his secon#ary anti*o#y often has an en-yme attache# to it0
,hich has a negligi*le effect on the *in#ing properties of the anti*o#y.
( su*strate for this en-yme is then a##e#. /ften0 this su*strate changes color upon
reaction ,ith the en-yme. $he color change sho,s that secon#ary anti*o#y has *oun# to
primary anti*o#y0 ,hich strongly implies that the #onor has ha# an immune reaction to
the test antigen. $his can *e helpful in a clinical setting0 an# in R)D.
$he higher the concentration of the primary anti*o#y that ,as present in the serum0 the
stronger the color change. /ften a spectrometer is use# to gi.e 8uantitati.e .alues for
color strength.
$he en-yme acts as an amplifier5 e.en if only fe, en-yme3linke# anti*o#ies remain *oun#0 the
en-yme molecules ,ill pro#uce many signal molecules. Kithin common3sense limitations the
en-yme can go on pro#ucing color in#efinitely0 *ut the more primary anti*o#y is present in the
#onor serum0 the more secon#ary anti*o#y G en-yme ,ill *in#0 an# the faster color ,ill #e.elop.
( major #isa#.antage of the in#irect E!'S( is that the metho# of antigen immo*ili-ation is non3
specific5 ,hen serum is use# as the source of test antigen0 all proteins in the sample may stick to
the microtiter plate ,ell0 so small concentrations of analyte in serum must compete ,ith other
serum proteins ,hen *in#ing to the ,ell surface. $he san#,ich or #irect E!'S( pro.i#es a
solution to this pro*lem0 *y using a 4capture4 anti*o#y specific for the test antigen to pull it out
of the serumFs molecular mi1ture.
E!'S( may *e run in a 8ualitati.e or 8uantitati.e format. Rualitati.e results pro.i#e a simple
positi.e or negati.e result yes or no" for a sample. $he cutoff *et,een positi.e an# negati.e is
#etermine# *y the analyst an# may *e statistical. $,o or three times the stan#ar# #e.iation error
inherent in a test" is often use# to #istinguish positi.e from negati.e samples. 'n 8uantitati.e
E!'S(0 the optical #ensity /D" of the sample is compare# to a stan#ar# cur.e0 ,hich is
typically a serial #ilution of a kno,n3concentration solution of the target molecule. For e1ample
if your test sample returns an /D of <.B0 the point on your stan#ar# cur.e that ga.e /D C <.B
must *e of the same analyte concentration as your sample.
San!:ic1 ELISA
A "an!:ic1 ELISA. <" :late is coate# ,ith a capture anti*o#y5 I" sample is a##e#0 an# any
antigen present *in#s to capture anti*o#y5 %" #etecting anti*o#y is a##e#0 an# *in#s to antigen5
@" en-yme3linke# secon#ary anti*o#y is a##e#0 an# *in#s to #etecting anti*o#y5 L" su*strate is
a##e#0 an# is con.erte# *y en-yme to #etecta*le form.
( less3common .ariant of this techni8ue0 calle# 4san#,ich4 E!'S(0 is use# to #etect sample
antigen. $he steps are as follo,s:
<. :repare a surface to ,hich a kno,n 8uantity of capture anti*o#y is *oun#.
I. Block any non specific *in#ing sites on the surface.
%. (pply the antigen3containing sample to the plate.
@. Kash the plate0 so that un*oun# antigen is remo.e#.
L. (pply en-yme linke# primary anti*o#ies as #etection anti*o#ies ,hich also *in#
specifically to the antigen.
E. Kash the plate0 so that the un*oun# anti*o#y3en-yme conjugates are remo.e#.
?. (pply a chemical ,hich is con.erte# *y the en-yme into a color or fluorescent or
electrochemical signal.
=. Measure the a*sor*ency or fluorescence or electrochemical signal e.g.0 current" of the
plate ,ells to #etermine the presence an# 8uantity of antigen.
$he image to the right inclu#es the use of a secon#ary anti*o#y conjugate# to an en-yme0
though technically this is not necessary if the primary anti*o#y is conjugate# to an en-yme.
2o,e.er0 use of a secon#ary3anti*o#y conjugate a.oi#s the e1pensi.e process of creating
en-yme3linke# anti*o#ies for e.ery antigen one might ,ant to #etect. By using an en-yme3
linke# anti*o#y that *in#s the Fc region of other anti*o#ies0 this same en-yme3linke# anti*o#y
can *e use# in a .ariety of situations. Kithout the first layer of 4capture4 anti*o#y0 any proteins
in the sample inclu#ing serum proteins" may competiti.ely a#sor* to the plate surface0 lo,ering
the 8uantity of antigen immo*ili-e#.9se of the purifie# specific anti*o#y to attach the antigen to
the plastic eliminates a nee# to purify the antigen from complicate# mi1tures *efore the
measurement0 simplifying the assay0 an# increasing the specificity an# the sensiti.ity of the
assay.
A#ication o* "an!:ic1 ELISA to 1ome $egnancy te"ting u"ing 1CG 1o$mone anti%o!y3
Cometiti'e ELISA
( thir# use of E!'S( is through competiti.e *in#ing. $he steps for this E!'S( are some,hat
#ifferent than the first t,o e1amples:
<. 9nla*ele# anti*o#y is incu*ate# in the presence of its antigen Sample"
I. $hese *oun# anti*o#y+antigen comple1es are then a##e# to an antigen coate# ,ell.
%. $he plate is ,ashe#0 so that un*oun# anti*o#y is remo.e#. $he more antigen in the
sample0 the less anti*o#y ,ill *e a*le to *in# to the antigen in the ,ell0 hence
4competition.4"
@. $he secon#ary anti*o#y0 specific to the primary anti*o#y is a##e#. $his secon# anti*o#y
is couple# to the en-yme.
L. ( su*strate is a##e#0 an# remaining en-ymes elicit a chromogenic or fluorescent signal.
For competiti.e E!'S(0 the higher the sample antigen concentration0 the ,eaker the e.entual
signal. $he major a#.antage of a competiti.e E!'S( is the a*ility to use cru#e or impure
samples an# still selecti.ely *in# any antigen that may *e present.
Note that some competiti.e E!'S( kits inclu#e en-yme3linke# antigen rather than en-yme3
linke# anti*o#y. $he la*ele# antigen competes for primary anti*o#y *in#ing sites ,ith your
sample antigen unla*ele#". $he more antigen in the sample0 the less la*ele# antigen is retaine#
in the ,ell an# the ,eaker the signal".Commonly the antigen is not first positione# in the ,ell.
Mu#ti#e an! +o$ta%#e ELISA (M<+ ELISA)
( ne, techni8ue E: < @>> =>@ B< in E:/ Bulletin IL.BI.IB> N. IBB>+B>5 9S:$/ ?L<BE=? in
9S:$/ Bulletin %<.B%.IBB>5 f! B%=<BBI>.B in S':/ :RC Bulletin B=.B@.IBB>" uses a soli#
phase ma#e up of an immunosor*ent polystyrene ro# ,ith =3<I protru#ing ogi.es. $he entire
#e.ice is immerse# in a test tu*e containing the collecte# sample an# the follo,ing steps
,ashing0 incu*ation in conjugate an# incu*ation in chromogenous" are carrie# out *y #ipping
the ogi.es in micro,ells of stan#ar# microplates pre3fille# ,ith reagents.
$he a#.antages of this techni8ue are as follo,s:
<. $he ogi.es can each *e sensiti-e# to a #ifferent reagent0 allo,ing the simultaneous
#etection of #ifferent anti*o#ies an# + or #ifferent antigens for multi3target assays5
I. $he sample .olume can *e increase# to impro.e the test sensiti.ity in clinical sali.a0
urine"0 foo# *ulk milk0 poole# eggs" an# en.ironmental ,ater" samples5
%. /ne ogi.e is left unsensiti-e# to measure the non3specific reactions of the sample5
@. $he use of la*oratory supplies for #ispensing sample ali8uots0 ,ashing solution an#
reagents in micro,ells is not re8uire#0 facilitating the #e.eopment of rea#y3to3use la*3
kits an# on3site kits.
-a!ioimmunoa""ay
-a!ioimmunoa""ay R'(" is an in .itro nuclear me#icine .ery sensiti.e techni8ue use# to
measure concentrations of antigens for e1ample0 hormone le.els in the *loo#" ,ithout the nee#
to use a *ioassay.
(lthough the R'( techni8ue is e1tremely sensiti.e an# e1tremely specific0 it re8uires speciali-e#
e8uipment0 *ut remains the least e1pensi.e metho# to perform such tests. 't re8uires special
precautions an# licensing0 since ra#ioacti.e su*stances are use#. $o#ay it has *een supplante#
*y the E!'S( metho#0 ,here the antigen3anti*o#y reaction is measure# using colorimetric
signals instea# of a ra#ioacti.e signal. 2o,e.er0 *ecause of its ro*ustness0 consistent results an#
lo, price per test 0 R'( metho#s are again *ecoming popular.
$he R(S$ test ra#ioallergosor*ent test" is an e1ample of ra#ioimmunoassay. 't is use# to #etect
the causati.e allergen for an allergy.
(ethod
$o perform a ra#ioimmunoassay0 a kno,n 8uantity of an antigen is ma#e ra#ioacti.e0 fre8uently
*y la*eling it ,ith gamma3ra#ioacti.e isotopes of io#ine attache# to tyrosine. $his ra#iola*ele#
antigen is then mi1e# ,ith a kno,n amount of anti*o#y for that antigen0 an# as a result0 the t,o
chemically *in# to one another. $hen0 a sample of serum from a patient containing an unkno,n
8uantity of that same antigen is a##e#. $his causes the unla*ele# or 4col#4" antigen from the
serum to compete ,ith the ra#iola*ele# antigen 4hot4" for anti*o#y *in#ing sites. (s the
concentration of 4col#4 antigen is increase#0 more of it *in#s to the anti*o#y0 #isplacing the
ra#iola*ele# .ariant0 an# re#ucing the ratio of anti*o#y3*oun# ra#iola*ele# antigen to free
ra#iola*ele# antigen. $he *oun# antigens are then separate# from the un*oun# ones0 an# the
ra#ioacti.ity of the free antigen remaining in the supernatant is measure# using a gamma
counter. 9sing kno,n stan#ar#s0 a *in#ing cur.e can then *e generate# ,hich allo,s the
amount of antigen in the patientFs serum to *e #eri.e#.
History
't ,as #e.elope# *y Rosalyn Jalo, an# Solomon (aron Berson in the <>LBs. 'n <>??0 Rosalyn
Sussman Jalo, recei.e# the No*el :ri-e in Me#icine for the #e.elopment of the R'( for
insulin: the precise measurement of minute amounts of such a hormone ,as consi#ere# a
*reakthrough in en#ocrinology.
Kith this techni8ue0 separating *oun# from un*oun# antigen is crucial. 'nitially0 the metho# of
separation employe# ,as the use of a secon# 4anti3anti*o#y4 #irecte# against the first for
precipitation an# centrifugation. $he use of charcoal suspension for precipitation ,as e1ten#e#
*ut replace# later *y Drs. Kerner an# (ce*e#o at Colum*ia 9ni.ersity for R'( of $% an# $@.
(n ultramicro R'( for human $S2 ,as pu*lishe# in BBRC <>?L" *y Drs. (ce*e#o0 2ayek et
al.
6%7
A#ication" o* -a!ioimmunoa""ay"
En!oc$ino#ogy
In"u#inE HCGE 7a"o$e""in
4etect" En!oc$ine 4i"o$!e$"
+1y"io#ogy o* En!oc$ine ,unction
+1a$maco#ogy
Mo$1ine
4etect 4$ug A%u"e o$ 4$ug +oi"oning
Stu!y 4$ug Hinetic"
Non i"otoic met1o!" o* !etection o* antigen"
Sen"iti'e non6i"otoic 4NA 1y%$i!i"ation a""ay o$ imme!iate6ea$#y antigen !etection *o$
$ai! i!enti*ication o* 1uman cytomega#o'i$u" in u$ine3
( sensiti.e non3ra#ioacti.e DN( hy*ri#isation assay employing #igo1igenin3la*elle# pro*es
,as compare# ,ith imme#iate3early antigen #etection an# con.entional .irus isolation for the
i#entification of human cytomegalo.irus 2CM&" in I@> urine samples. /f @@ specimens
yiel#ing 2CM& *y .irus isolation0 more ,ere positi.e *y DN( hy*ri#isation %I5 ?%Z" than *y
imme#iate3early antigen #etection IL5 LIZ" : C B.BL". $he specificity of the hy*ri#isation
assay in @L apparently falsely positi.e specimens ,as supporte# *y #etection of 2CM& DN( in
@B of these specimens using the polymerase chain reaction. Many urine specimens may thus
contain large amounts of non3.ia*le .irus or free .iral DN(. E.aluation of .arious protocols for
the e1traction an# #enaturation of .irus DN( prior to hy*ri#isation sho,e# that proteinase N
#igestion ,ith phenol+chloroform e1traction ,as the most sensiti.e an# relia*le proce#ure. Ke
conclu#e that the non3ra#ioacti.e DN( hy*ri#isation assay #escri*e# is a potentially .alua*le
routine #iagnostic test.
Cytomega#o'i$u" !etection %y noni"otoic in "itu 4NA 1y%$i!i;ation an! 'i$a# antigen
immuno"taining u"ing a t:o6co#o$ tec1ni.ue3
Rapi# metho#s of specific .iral #iagnosis in formalin fi1e#0 paraffin em*e##e# tissues inclu#e
i#entification of .iral incusions in routinely staine# histologic sections0 immunologic staining of
.iral antigens0 an# in situ nucleic aci# hy*ri#i-ation. $o correlate in situ hy*ri#i-ation ,ith
immunologic #etection metho#s0 se8uential t,o3color staining ,as use# on tissues from <I
patients0 each containing characteristic cytomegalo.irus CM&" inclusions0 using a *iotinylate#
CM& DN( pro*e in an a.i#in3alkaline phosphatase3linke# reaction follo,e# *y a.i#in3*iotin
comple1 immunopero1i#ase staining of CM& antigen. CM& genetic material ,as seen in all <?
tissues. CM& antigen ,as #etecte# in << of <? tissues ELZ". $he DN( hy*ri#i-ation techni8ue
pro.i#e# more intense staining0 #etecte# greater num*ers of inclusions0 an# ha# less *ackgroun#
staining than the immunopero1i#ase techni8ue. $he alkaline phosphatase reaction pro#uct ,as
sta*le through su*se8uent immunostaining steps0 an# immunologic reacti.ity of CM& antigen
,as not significantly re#uce# *y prior hy*ri#i-ation steps. CM& DN( pro*e ,as locali-e#
pre#ominantly ,ithin cell nuclei0 ,hile CM& antigen immunostaining ,as pre#ominantly
cytoplasmic. 't ,as conclu#e# that se8uential in situ hy*ri#i-ation an# immunocytochemistry
can *e performe# on stan#ar# histologic sections. Furthermore0 it is likely that the majority of
CM& nucleic aci# #etecte# *y this tissue hy*ri#i-ation techni8ue is unencapsi#ate#0 intranuclear
.iral DN( an# not DN( containe# ,ithin complete CM& nucleocapsi#s.

C1emi#umine"cence
C1emi#umine"cence sometimes 4c1emo#umine"cence4" is the emission of light ,ith limite#
emission of heat luminescence"0 as the result of a chemical reaction. ;i.en reactants A an# B0
,ith an e1cite# interme#iate I0
6A7 G 6B7 g 6I7 g 6Products7 G light
For e1ample0 if 6(7 is luminol an# 6B7 is hy#rogen pero1i#e in the presence of a suita*le catalyst
,e ha.e:
luminol G 2
I
/
I
g %3(:(6I7 g %3(:( G light
,here:
,here %3(:( is %3aminophthalate
%3(:(6I7 is the e1cite# state fluorescing as it #ecays to a lo,er energy le.el.
$he #ecay of the e1cite# state6I7 to a lo,er energy le.el causes the emission of light. 'n theory0
one photon of light shoul# *e gi.en off for each molecule of reactant0 or (.oga#roFs num*er of
photons per mole. 'n actual practice0 non3en-ymatic reactions sel#om e1cee# <Z R
C
0 8uantum
efficiency.
'n a chemical reaction0 reactants colli#e to form a transition state0 the enthalpic ma1imum in a
reaction coor#inate #iagram0 ,hich procee#s to the pro#uct. Normally0 reactants form pro#ucts
of lesser chemical energy. $he #ifference in energy *et,een reactants an# pro#ucts0 represente#
as h&
r'n
0 is turne# into heat0 physically reali-e# as e1citations in the .i*rational state of the
normal mo#es of the pro#uct. Since .i*rational energy is generally much greater than the
thermal agitation0 it is rapi#ly #isperse# into the sol.ent through sol.ent moleculesF rotation an#
translation. $his is ho, e1othermic reactions make their solutions hotter. 'n a chemiluminescent
reaction0 the #irect pro#uct of a reaction is #eli.ere# in an e1cite# electronic state0 ,hich then
#ecays into an electronic groun# state through either fluorescence or phosphorescence0
#epen#ing on the spin state of the electronic e1cite# state forme#. $his is possi*le *ecause
chemical *on# formation can occur on a timescale faster than electronic transitions0 an#
therefore can result in #iscrete pro#ucts in e1cite# electronic states.
Chemiluminescence #iffers from fluorescence in that the electronic e1cite# state is #eri.e# from
the pro#uct of a chemical reaction rather than the more typical ,ay of creating electronic e1cite#
states0 namely a*sorption. 't is the antithesis of a photochemical reaction0 in ,hich light is use#
to #ri.e an en#othermic chemical reaction. 2ere0 light is generated from a chemically
e1othermic reaction.
( stan#ar# e1ample of chemiluminescence in the la*oratory setting is foun# in the luminol test0
,here e.i#ence of *loo# is taken ,hen the sample glo,s upon contact ,ith iron. Khen
chemiluminescence takes place in li.ing organisms0 the phenomenon is calle# *ioluminescence.
( lightstick emits light *y chemiluminescence.
0i1uid"phase reactions
!uminol in an alkaline solution ,ith hy#rogen pero1i#e in the presence of iron or
copper0 or an au1iliary o1i#ant0 pro#uces chemiluminescence. $he luminol reaction is
luminol G 2
I
/
I
g %3(:(6I7 g %3(:( G light
$he 8uantum efficiency0 R
C
is <Z. For the la*oratory e1periment see references.
Cyalume0 as use# in a lightstick0 emits light *y chemiluminescence of a fluorescent #ye
also calle# fluorescor" acti.ate# *y cyalume reacting ,ith hy#rogen pero1i#e in the
presence of a catalyst0such as so#ium salicylate. 't is the most efficient chemiluminescent
reaction kno,n. up to <LZ 8uantum efficiency.
cyalume G 2
I
/
I
G #ye g phenol G IC/
I
G #ye6I7
Khen the acti.ate# fluorescent #ye #ecays to a lo,er energy le.el0 light is gi.en off. $he color
#epen#s upon the #ye.
Co#o$ Sen"iti"e$
Blue >0<B3Diphenylanthracene
;reen >0<B3Bisphenylethynyl"anthracene
Jello,3green $etracene
Jello, <3Chloro3>0<B3*isphenylethynyl"anthracene
/range L0<I3Bisphenylethynyl"naphthacene0 Ru*rene0 Rho#amine E;
Re# Rho#amine B
/1alyl chlori#e C
I
/
I
Cl
I
" pro#uces light ,hen o1i#i-e# 3 *ut only in the presence of a
sensitiser0 similar to the a*o.e e1amples. 'f o1alyl chlori#e is treate# ,ith 2
I
/
I
in non-
a(ueous me#ia e.g. C2
I
Cl
I
" in the presence of a sensitiser0 emission of light is o*taine#.
$he colour0 intensity an# #uration of light emission #epen# on the sensitiser use#.
Ro#amin E ; gi.es *right orange light ,ith mo#erate #uration of emission.
Ru*ipy"
%
IG
is a ruthenium''" comple1 ,hich un#ergoes o1i#ation to ruthenium'''" if
certain o1i#i-ing agents are intro#uce#. 'f ruthenium'''" comple1 is then re#uce# in
alkaline me#ium0 emission of light occurs. First0 there is a reaction:
IRu*ipy"
%
IG
G :*/
I
G @2
G
g IRu*ipy"
%
%G
G :*
IG
G I2
I
/
2ere0 Ru'''" is o*taine#. Further reaction inclu#es use of solution of so#ium
tetrahy#ro*orate'''"0 NaB2
@
in alkaline me#ium. Khen the solution is a##e#0 Ru'''" is
re#uce# to Ru''" an# orange light is emitte#.
$M(E tetrakis#imethylamino"ethylene" emits clear *lue3green light upon o1i#ation *y
air.
:yrogallol <0I0%3trihy#ro1i*en-ene" is also capa*le of light emission. 'f an a8ueous
solution of pyrogallol0 Na/2 an# N
I
C/
%
is mi1e# ,ith formal#ehy#e0 short3li.e# re#
emission occurs.
Singlet o1ygen /
I
" can also emit light. 'f solutions of %BZ hy#rogen pero1i#e an# LZ
alkaline so#ium hypochlorite NaCl/" are mi1e#0 re# light is emitte#. 't is *arely .isi*le0
though 3 for this reason a sensitiser is often inclu#e# to *oost light emission in terms of
*rightness an# intensity. (gain0 *oth colour an# intensity of light #epen# on the sensitiser
use#.
!ucigenin o1i#ation is also .ery ,ell kno,n among chemiluminescence reactions. 'f an
a8ueous lucigenin solution is mi1e# ,ith highly alkaline a8ueous solution containing
ethanol or acetone an# hy#rogen pero1i#e0 .ery *right green emission is pro#uce# that
#ecays to greenish *lue an# finally *lue emission. $he #uration of the emission can *e up
to a couple of minutes un#er the right circumstances.
Solutions containing ions of manganese &''0 '&0 '''" sho, chemiluminescence E>B nm"
,hen re#uce# *y so#ium *orohy#ri#e at lo, p2 to Mn''"
/ther li8ui# phase chemilumiscence reagents:
o pero1yo1alates
o (ryl o1alates
o (cri#iniumesters
o #io1etanes
#as"phase reactions
( green an# *lue glo,stick.
/ne of the ol#est kno,n chemoluminescent reactions is that of elemental ,hite
phosphorus o1i#i-ing in moist air0 pro#ucing a green glo,. $his is a gas3phase reaction
of phosphorus .apor0 a*o.e the soli#0 ,ith o1ygen pro#ucing the e1cite# states :/"
I
an#
2:/.
(nother gas phase reaction is the *asis of nitric o1i#e #etection in commercial analytic
instruments applie# to en.ironmental air 8uality testing. /-one is com*ine# ,ith nitric
o1i#e to form nitrogen #io1i#e in an acti.ate# state.
N/G/
%
g N/
I
6I7G /
I

$he acti.ate# N/
I
6I7 luminesces *roa#*an# .isi*le to infrare# light as it re.erts to a
lo,er energy state. ( photomultiplier an# associate# electronics counts the photons
,hich are proportional to the amount of N/ present. $o #etermine the amount of
nitrogen #io1i#e0 N/
I
0 in a sample containing no N/" it must first *e con.erte# to nitric
o1i#e0 N/0 *y passing the sample through a con.erter *efore the a*o.e o-one acti.ation
reaction is applie#. $he o-one reaction pro#uces a photon count proportional to N/
,hich is proportional to N/
I
*efore it ,as con.erte# to N/. 'n the case of a mi1e#
sample containing *oth N/ an# N/
I
0 the a*o.e reaction yiel#s the amount of N/ an#
N/
I
com*ine# in the air sample0 assuming that the sample is passe# through the
con.erter. 'f the mi1e# sample is not passe# through the con.erter0 the o-one reaction
pro#uces acti.ate# N/
I
6I7 only in proportion to the N/ in the sample. $he N/
I
in the
sample is not acti.ate# *y the o-one reaction. $hough unacti.ate# N/
I
is present ,ith
the acti.ate# N/
I
6I70 photons are only emitte# *y the acti.ate# species ,hich is
proportional to original N/. Final step0 su*tract N/ from N/ G N/
I
" to yiel# N/
I

$nhanced chemiluminescence
En1ance! c1emi#umine"cence is a common techni8ue for a .ariety of #etection assays in
*iology. ( horsera#ish pero1i#ase en-yme 2R:" is tethere# to the molecule of interest usually
through la*eling an immunoglo*ulin that specifically recogni-es the molecule". $his en-yme
comple10 then cataly-es the con.ersion of the enhance# chemiluminescent su*strate into a
sensiti-e# reagent in the .icinity of the molecule of interest0 ,hich on further o1i#ation *y
hy#rogen pero1i#e0 pro#uces a triplet e1cite#" car*onyl ,hich emits light ,hen it #ecays to the
singlet car*onyl. Enhance# chemiluminescence allo,s #etection of minute 8uantities of a
*iomolecule. :roteins can *e #etecte# #o,n to femtomole 8uantities0 ,ell *elo, the #etection
limit for most assay systems.
Applications
gas analysis: for #etermining small amounts of impurities or poisons in air. /ther
compoun#s can also *e #etermine# *y this metho# o-one0 N3o1i#es0 S3compoun#s". (
typical e1ample is N/ #etermination ,ith #etection limits #o,n to < pp*
analysis of inorganic species in li8ui# phase
analysis of organic species: useful ,ith en-ymes0 ,here the su*strate isnFt #irectly
in.ol.e# in chemiluminescence reaction0 *ut the pro#uct is
#etection an# assay of *iomolecules in systems such as E!'S( an# Kestern *lots
DN( se8uencing using pyrose8uencing
!ighting o*jects. Chemiluminescence kites0 emergency lighting0 glo, sticks party
#ecorations".
Com*ustion analysis: certain ra#ical species such as C2i an# /2i" gi.e off ra#iation at
specific ,a.elengths. $he heat release rate is calculate# *y measuring the amount of
light ra#iate# from a flame at those ,a.elengths.
chil#renFs toys
E#ect$oc1emi#umine"cence
E#ect$oc1emi#umine"cence or e#ect$ogene$ate! c1emi#umine"cence ECL" is a kin# of
luminescence pro#uce# #uring electrochemical reactions in solutions. 'n electrogenerate#
chemiluminescence0 electrochemically generate# interme#iates un#ergo a highly e1ergonic
reaction to pro#uce an electronically e1cite# state that then emits light. EC! e1citation is cause#
*y energetic electron transfer re#o1" reactions of electrogenerate# species. Such luminescence
e1citation is a form of chemiluminescence ,here one+all reactants are pro#uce#
electrochemically on the electro#es.
EC! is usually o*ser.e# #uring application of potential se.eral .olts" to electro#es of
electrochemical cell that contains solution of luminescent species polycyclic aromatic
hy#rocar*ons0 metal comple1es" in aprotic organic sol.ent EC! composition".
Application
EC! pro.e# to *e .ery useful in analytical applications as a highly sensiti.e an# selecti.e
metho#. 't com*ines analytical a#.antages of chemiluminescent analysis a*sence of
*ackgroun# optical signal" ,ith ease of reaction control *y applying electro#e potential.
Enhance# selecti.ity of EC! analysis is reache# *y .ariation of electro#e potential thus
controlling species that are o1i#i-e#+re#uce# at the electro#e an# take part in EC! reaction.
't generally uses Ruthenium comple1es0 esp 6Ru Bpy"%7
IG
,hich releases a photon at cEIB nm"
regenerating ,ith $:( $ripropylamine" in li8ui# phase or li8ui#3soli# interface. 't can *e use#
as monolayer immo*ili-e# on an electro#e surface ma#e eg of nafion0 or special thin films
ma#e *y !angmuir3Blogett techni8ue or self3assem*ly techni8ue" or as a coreactant or more
commonly as a tag an# use# in 2:!C0 Ru tagge# anti*o#y *ase# immunoassays0 Ru $agge#
DN( pro*es for :CR etc0 N(D2 or 2
I
/
I
generation *ase# *iosensors0 o1alate an# organic
amine #etection an# many other applications an# can *e #etecte# from picomolar sensiti.ity to
#ynamic range of more than si1 or#ers of magnitu#e. :hoton #etection is #one ,ith
photomultiplier tu*es :M$" or silicon photo#io#e or gol# coate# fi*er3optic sensors. EC! is
hea.ily use# commercially for many clinical la* applications.
UNIT III ASSESSMENT O, CELL ME4IATE4 IMMUNITY
I!enti*ication o* #ym1ocyte" an! t1ei$ "u%"et" in %#oo!
$o clarify the immune mechanism in myocar#itis0 immunofluorescence

techni8ues ,ith laser
flo, cytometry ,ere use# to e1amine serial changes in

lymphocyte su*sets in the heart0 spleen0
an# peripheral *loo# of DB(+I an#

B(!B+c mice inoculate# ,ith encephalomyocar#itis .irus
E1periment '". B

cells ,ere i#entifie# *y staining ,ith fluorescein isothiocyanate3la*elle#

ra**it
anti3mouse immunoglo*ulin. $3 cell su*sets ,ere i#entifie# ,ith rat

anti3$hy <.I0 an#
nonpolymorphic !yt < an# !yt I monoclonal anti*o#ies plus

fluorescein isothiocyanate3 la*elle#
anti3mouse immunoglo*ulin. /n #ays ?

an# <@ postinfection0 the percentage of $hy <.IG pan $"
cells in *oth

strains ha# #ecrease# in the peripheral *loo#5 B cells sho,e# no

significant changes
throughout the entire perio#. /n the other han#0 $hy

<.IG pan $" an# !yt <G0 I%G precursor
an# immature" $ cells appeare# to

occupy the major portion of the myocar#ium on #ays ? an# <@
,hen congesti.e

heart failure #e.elope#. $o confirm this0 serial immunohistologic stu#ies
immunopero1i#ase staining" of the hearts of DB(+I an# B(!B+c mice ,ith
encephalomyocar#itis .irus3in#uce# myocar#itis ,ere performe# E1periment

''". 'n E1periment
''0 most of the staine# cells in the hearts of *oth

strains ,ere $hy <.I positi.e an# !yt < an# !yt
I positi.e on #ays ? an#

<@. $hus0 E1periments ' an# '' #emonstrate# that lymphocytes at the
site of

inflammation in acute .iral myocar#itis carrie# antigenic markers that

#iffere# from those
of peripheral lymphocytes an# suggeste# that $hy <.IG

pan $" cells0 especially the !yt <G0 I%G
su*set immature $ cells an#

$3cell su*set precursors" ,ere in.ol.e# in the #e.elopment of
myocar#itis

in these animals.
T ce## acti'ation a$amete$"
?3 T #ym1ocyte acti'ation gene i!enti*ication %y co$egu#ate! e&$e""ion on 4NA m
c$oa$$ay"3
2igh3capacity metho#s for assessing gene function ha.e *ecome increasingly important *ecause
of the increasing num*er of ne,ly i#entifie# genes emerging from large3scale genome
se8uencing an# cDN( cloning efforts. Ke in.estigate# the use of DN( microarrays to i#entify
uncharacteri-e# genes specifically in.ol.e# in human $ cell acti.ation. (cti.ation of human
peripheral *loo# $ lymphocytes in#uce# significant changes in hun#re#s of transcripts0 *ut most
of these ,ere not uni8ue to $ cell acti.ation. &ariation of e1perimental parameters an# analysis
techni8ues allo,e# *etter enrichment for gene e1pression changes uni8ue to $ cell acti.ation.
Best results ,ere achie.e# *y i#entification of genes that ,ere most highly coregulate# ,ith the
$3cell3specific transcript interleukin I '!I" in a 4compen#ium4 of e1periments in.ol.ing *oth $
cells an# other cell types. (mong the genes most highly coregulate# ,ith '!I ,ere many genes
kno,n to function #uring $ cell acti.ation0 together ,ith ES$s of unkno,n function. Four of
these ES$s ,ere e1ten#e# to no.el full3length clones enco#ing $3cell3regulate# proteins ,ith
pre#icte# functions in ;$: meta*olism0 cell organi-ation0 an# signal trans#uction.
23 4ete$mination o* "o#u%#e C42? a" a a$amete$ o* B ce## acti'ation3
'n this stu#y ,e esta*lishe# a no.el soli#3phase immunoassay for CDI< using the time3resol.e#
fluorescence of lanthani#e chelates. $he capture assay ,as a*le to #etect concentrations of as
lo, as <BB pg of CDI< antigen per millilitre of sample an# ,as use# for 8uantitati.e
#etermination of CDI< in lysates of #ifferent cell lines as ,ell as in patient serum specimens.
CDI< ,as measure# in lysates of tonsils an# cell lines of B0 $ cell an# myelomonocyte lineage0
an# appeare# to consist of monomeric antigen un#er the #etergent con#itions use#. Ele.ate#
le.els of solu*le CDI< ,ere o*ser.e# in serum of patients ,ith Epstein3Barr .irus EB&"
infection0 a #isease kno,n to *e associate# ,ith polyclonal B cell acti.ation0 an# in infection
,ith the lymphotropic ru*ella .irus. Significantly increase# le.els ,ere also foun# in
malignancies ,hich are associate# ,ith EB&. 'n patients ,ith nasopharyngeal carcinoma N:C"0
a correlation ,ith the titre of EB&3specific 'g( ,as o*ser.e#0 thus supporting a possi*le role of
solu*le CDI< as a marker for #isease acti.ity in certain malignancies. /ur #ata suggest that
measurement of solu*le CDI< coul# ser.e as a marker for acti.ation of the immune system an#
#iseases in.ol.ing the B cell lymphoi# system. :ossi*le mechanisms an# functions of solu*le
CDI< are #iscusse#.
@3 Mu#ti#e C44 an! C4D T6ce## acti'ation a$amete$" $e!ict 'accine e**icacy in vivo
me!iate! %y in!i'i!ua# 4C6acti'ating agoni"t"
( systematic comparison of the immunostimulatory capacity of $!R I0 %0 @0 L0 ? an# > agonists
an# an agonistic CD@B3specific anti*o#y ,as performe# in a single long pepti#e .accination
mo#el. (ll a#ju.ants acti.ate# DC in !itro *ut not all in#uce# a strong functional $3cell response
in !i!o. /ptimal clonal CD=G $3cell e1pansion #epen#e# on the capacity of agonists to mature
pro3inflammatory DC an# the #uration of their in !i!o stimulatory effect. Strong agonists
promote# the in#uction of *oth antigen3specific 'FN^3pro#ucing CD@G $3helper cells an# high
num*ers of 'FN^ pro#ucing CD=G effector $3cells that kille# target cells in !i!o. 'mportantly0 the
capacity of an agonist to function as an a#ju.ant #epen#e# on the .accine strategy use#.
Collecti.ely0 the multi3parameter system presente# here can *e use# as a general roa# map to
#e.elop therapeutic .accines.
43 Anti68a626in!uce! T ce## acti'ation3 T1e a$amete$" o* acti'ationE t1e !e*inition o*
mitogenic an! nonmitogenic anti%o!ie"E an! t1e !i**e$entia# e**ect" on C44J '" C4DJ T
ce##"
$he M2C (g Ra3I is a glycolipi# anchore# class ' molecule e1presse# at high le.els on all
peripheral $ lymphocytes. 'n this stu#y ,e foun# that anti3Ra3I anti*o#ies coul# stimulate the
proliferation of murine $ cells in .itro. (nti3Ra3I3in#uce# proliferation re8uire# secon#ary
cross3linking ,ith anti3'g anti*o#y an# the presence of :M(. /nly Ra3IG strains coul# *e
in#uce# to proliferate *y anti3Ra3I anti*o#y0 *ut un#er the con#itions employe#0 anti3CD% coul#
in#uce proliferation in Ra3IG an# Ra3I3strains. 'nterestingly0 only anti3Ra3I reagents #irecte#
against the alpha % #omain of the Ra3I class ' molecule ,ere effecti.e in in#ucing proliferation.
Furthermore0 unlike purifie# CD@G cells0 purifie# CD=G cells ,ere una*le to *e stimulate# *y
the anti3Ra3I anti*o#ies. $hese results lea# to the inclusion of Ra3I in a group of
physiologically rele.ant0 glycolipi#3anchore#0 cell3surface molecules0 mo*ili-ation of ,hich can
generate signals that initiate the proliferation of $ cells. Such molecules may play a secon#ary
role in cellular acti.ation after the primary engagement of the $CR.
B3 -e"i"tance to mac$o1age6me!iate! 5i##ing a" a *acto$ in*#uencing t1e at1ogene"i" o*
c1$onic cutaneou" #ei"1mania"i"
Cutaneous leishmaniasis can *e either a spontaneously healing or chronic #isease0 #epen#ing
upon the strain of parasite an# the immunological status of the host. Ke ha.e in.estigate#
parasite factors responsi*le for the .aria*le pathogenesis o*ser.e# in leishmanial infections *y
testing the sensiti.ity of se.eral leishmanial strains to intracellular killing in lymphokine !N"
acti.ate# mouse macrophages. Significant micro*ici#al acti.ity against !eishmania tropica0 a
strain ,hich heals in CL?B!+E BE" mice0 ,as foun#. 'n contrast0 a strain Maria" ,hich has
pre.iously *een sho,n to in#uce chronic nonhealing cutaneous lesions in BE mice ,as resistant
to killing in acti.ate# macrophages. $his resistance to killing ,as o*ser.e# in macrophages
acti.ate# *y !N o*taine# from either Bacille Calmette3;uerin30 !. tropica0 or the Maria strain
infecte# mice. $he ina*ility of !N acti.ate# macrophages to kill the Maria strain ,as sho,n not
to *e #ue to parasite in#uce# inhi*ition of killing mechanisms0 since Maria strain infecte#0 !N
treate# macrophages e1hi*ite# tumorici#ial acti.ity similar to uninfecte# macrophages.
Furthermore0 !N acti.ate# macrophages simultaneously infecte# ,ith the Maria strain an#
another intracellular pathogen0 $o1oplasma gon#ii0 kille# $o1oplasma0 *ut not the Maria strain.
$emperature ,as also foun# to significantly influence the multiplication an# killing of
!eishmania parasites. (s ,oul# *e e1pecte# from their cutaneous nature0 !. tropica an# Maria
strain parasites multiplie# *etter at %L #egrees C than at %? #egrees C. (lso consistent ,ith the
failure of cutaneous strains to .iscerali-e in immunocompetent mice ,as the o*ser.ation that the
killing of leishmanial parasites ,as enhance# at the higher temperature. $hus0 the temperature
#epen#ent gro,th capacity an# sensiti.ity to killing of a gi.en leishmanial strain in
macrophages may *e important factors influencing the pathogenesis of cutaneous leishmaniasis.
63 E**ect o* atu#in on mic$o%ici!a# acti'ity o* mou"e e$itonea# mac$o1age"
:atulin0 a fungal meta*olite sho,n pre.iously to e1ert immunosuppressi.e effects on the cellular
an# humoral immune systems0 ,as e1amine# for its in !itro effects on some functions of murine
peritoneal macrophages. $he cells ,ere pre3incu*ate# for Ihr ,ith mycoto1in concentrations of
B.B<AI ag+ml. :hagocytosis an# phagosome3lysosome fusion ,ere #iminishe# a*o.e B.< ag
patulin+ml an# lysosomal n-ymes an# micro*ici#al acti.ity a*o.e B.L ag+ml0,hereas /
I
j
pro#uction ,as inhi*ite# only a*o.e I ag+ml. $his in#icate# that the killing mechanism #i# not
#epen# on pro#ucts of the o1i#ati.e *urst. $he concentrations use# #i# not #ecrease the cell
.ia*ility. 9n#er natural circumstances0 patulin may constitute a health risk for animals.
CYTOHINES
Cytokines are *elie.e# to *e in.ol.e# in the pathogenesis an# enhance# e1pression in patients
,ith 2o#gkinHs an# Non32o#gkins lymphoma. Base# on this phenomenon0 a multicentric stu#y
,as carrie# out in .arious lymphoma cases. $he #iagnosis of lymphoma ,as ma#e on tissue
*iopsies an# fine nee#le aspiration cytology FN(C". /ut of a total of ?I cases stu#ie#0 @L ,ere
of 2o#gkinHs lymphoma EI.LZ" an# I? cases ,ere of Non32o#gkinHs lymphoma %?.LZ".
Ma1imum cases of 2o#gkinHs #isease occurre# in the age group of %B3@B years an# males
outnum*ere# females. 2o#gkinHs lymphoma cases ,ere pre#ominantly of mi1e# cellularity
histologic type @E.EEZ" ,hereas majority cases of Non32o#gkinHs lymphoma ,ere of high
gra#e histologic type @=.<@Z" ,ith pre#ominance in the age group L<3EB years. 'n *oth these
type of lymphomas0 the '!3IR an# '!3E le.els ,ere foun# to *e increase# more than four fol#
as compare# to healthy controls" pMB.BL". $he cytokine le.els #ecrease# after chemotherapy in
patients sho,ing response to therapy. 2o,e.er0 there ,ere fe, conflicting an# unrelia*le tren#s
in the '!3E le.els after chemotherapy ,here ele.ate# '!3E le.els persiste# in patients in clinical
remission. /.erall0 it ,as seen that *oth '!3IR an# '!3 E can *e use# as an in#icator for
assessing prognosis an# #rug therapy in lymphoma cases. '!3IR ,as foun# to *e a *etter
prognostic marker than '!3E in assessing the response of lymphoma patient to chemotherapy0
more so in 2o#gkinHs #isease.
Mac$o1age Acti'ation
Macrophage effector function significantly influences the 8uality0 #uration0 an#
magnitu#e of most inflammatory reactions. $ra#itionally0 macrophages ha.e *een #escri*e# as
antigen3presenting phagocytes that secrete pro3inflammatory an# antimicro*ial me#iators.
Mounting e.i#ence0 ho,e.er0 #escri*es a more comple1 mo#el in.ol.ing multiple macrophage
phenotypes carrying out #ifferential functions an# eliciting #i.ergent effects on surroun#ing
cells an# tissues. Stein et al. ,ere the first to #escri*e 4alternati.ely4 acti.ate# macrophages as
ha.ing a phenotype #istinct from ,hat are no, calle# 4classically4 acti.ate# macrophages. From
this seminal o*ser.ation0 a mo#el of t,o major macrophage classes has #e.elope#. Classically
acti.ate# macrophages e1hi*it a $h<3like phenotype0 promoting inflammation0 e1tracellular
matri1 ECM" #estruction0 an# apoptosis0 ,hile alternati.ely acti.ate# macrophages #isplay a
$hI3like phenotype0 promoting ECM construction0 cell proliferation0 an# angiogenesis.
(lthough *oth phenotypes are important components of *oth the innate an# a#apti.e immune
systems0 the classically acti.ate# macrophage ten#s to elicit chronic inflammation an# tissue
injury ,hereas the alternati.ely acti.ate# macrophage ten#s to resol.e inflammation an#
facilitate ,oun# healing Figure <".
,igu$e ?3 Schematic of classical (" .ersus alternati.e B"
acti.ation of macrophages #epicting the priming signal classical
only" an# stimuli0 their effects on cellular function0 an#
su*se8uent effects on surroun#ing tissue physiology. 6Note: figure
a#apte# from ;or#on0 S. IBB%" Nat. Re.. 'mmunol. @:I%.7
C#a""ica##y Acti'ate! Mac$o1age"
Differentiation of classically acti.ate# macrophages re8uires a priming signal in the form of
'FN3gamma
?
.ia the 'FN3gamma R. Khen the prime# macrophage su*se8uently encounters an
appropriate stimulus0 such as *acterial !:S0 it *ecomes classically acti.ate#. !:S is first *oun#
*y solu*le !B: an# then *y either solu*le or mem*rane3*oun# CD<@. CD<@ #eli.ers !:S to the
!:S recognition comple10 ,hich consists of at least $!R@<B an# MD3I. :athogens an#
pathogen components are su*se8uently taken up *y phagocytosisan# #eli.ere# to lysosomes
,here they are e1pose# to a .ariety of #egra#ation en-ymes inclu#ing se.eral Cathepsin
cysteine proteases. Suita*le antigens are processe# an# loa#e# onto M2C class '' molecules in
late en#ocytic compartments an# antigen+M2C'' comple1es as ,ell as co3stimulatory B? family
mem*ers are presente# to $ cells.
$hese e.ents are follo,e# closely *y a significant change in cellular morph3ology an# a
#ramatic alteration in the secretory profile of the cell. ( .ariety of chemokines inclu#ing '!3
=+CXC!=0 ':3<B+CXC!<B0 M':3< alpha+CC!%0 M':3< *eta+CC!@0 an# R(N$ES+CC!L0 are
release# as chemoattractants for neutrophils0 immature #en#ritic cells0 natural killer cells0 an#
acti.ate# $ cells. Further0 se.eral pro3inflammatory cytokines are release# inclu#ing '!3<
*eta+'!3<FI0 '!3E0 an# $NF3alpha+$NFSF<(. $NF3alpha also contri*utes to the pro3apoptotic
acti.ity of the classically acti.ate# macrophage. $NF3alpha is accompanie# *y Fas
!igan#+$NFSFE secretion
<E
an# N/ release as a result of iN/S upregulation. 'n a##ition0 the
classically acti.ate# macrophage releases proteolytic en-ymes inclu#ing MM:3<0 3I0 3?0 3>0 an#
3<I0 ,hich #egra#e Collagen0 Elastin0 Fi*ronectin0 an# other ECM components.
Khile the release of these molecules is important for host #efense an# #irection of the a#apti.e
immune system0 ,hen uncontrolle# they can le.y significant collateral #amage on the
microen.ironment. By eliciting massi.e leukocyte infiltration an# floo#ing the surroun#ing
tissue ,ith inflammatory me#iators0 pro3apoptotic factors0 an# matri1 #egra#ing proteases0 the
classically acti.ate# macrophage is capa*le of #ismantling tissues to the point of inflicting
serious injury. $issue #estruction perpetrate# *y chronic inflammation has *een associate# ,ith
the #e.elopment of tumors0 type < autoimmune #iseases0 an# glomerulonephritis among other
pathologies Figure <(".
A#te$nati'e#y Acti'ate! Mac$o1age"
Differentiation of alternati.ely acti.ate# macrophages #oes not re8uire any priming. '!3@
I
an#+or '!3<% can act as sufficient stimuli. $he *in#ing of these factors to their respecti.e
receptors is follo,e# *y flui#3phase pinocytosis of solu*le antigen.Solu*le antigen is then
loa#e# onto M2C class '' molecules an# antigen+M2C'' comple1es an# co3stimulatory B?
family mem*ers are su*se8uently #isplaye# to $ cells.
Similar to the classically acti.ate# macrophage0 the alternati.ely acti.ate# macrophage changes
its cellular morphology an# secretory pattern as a result of appropriate stimulation. !eukocytes
are attracte# *y the macrophage .ia its release of chemokines inclu#ing
MDC+CC!II:(RC+CC!<=an# $(RC+CC!<?. 'nflammation is counteracte# *y the release of
factors such as '!3<ra+'!3<F%0 Jm<0 JmI0 RE!Ma0'!3<B0an# $;F3*eta. $;F3*eta also
functions in#irectly to promote ECM *uil#ing *y in#ucing near*y fi*ro*lasts to pro#uce ECM
components. $he alternati.ely acti.ate# macrophage itself secretes the ECM components0
Fi*ronectin an# *';32%0 the ECM cross3linking en-yme0 $rans3glutaminase0 an# /steopontin0
,hich is in.ol.e# in cell a#hesion to the ECM.
'n a##ition0 alternati.ely acti.ate# macrophages upregulate the en-yme (rginase '0 ,hich is
in.ol.e# in proline as ,ell as polyamine *iosynthesis. :roline promotes ECM construction
,hile polyamines are in.ol.e# in cell proliferation. /ther factors secrete# *y the alternati.ely
acti.ate# macrophage that promote cell proliferation inclu#e :D;F0 ';F0 an# $;F3*eta. $hese
factors0 along ,ith F;F *asic0 $;F3alpha0 an# &E;F0 also participate in angiogenesis.
$he molecules secrete# *y the alternati.ely acti.ate# macrophage ,ork to,ar# resolution of
inflammation an# promotion of ,oun# repair #ue to their anti3inflammatory0 fi*rotic0
proliferati.e0 an# angiogenic acti.ities. $his macrophage is also especially efficient at com*ating
parasitic infections such as Schistosomiasis. 'n a##ition to its *eneficial acti.ities0 the
alternati.ely acti.ate# macrophage has *een implicate# in se.eral pathologies0 the most
prominent of ,hich are allergy an# asthma Figure <B".
Mac$o1age Mic$o%ici!a# A""ay"
( main function of macrophages in the host #efense against infection is the phagocytosis of
nonopsoni-e# or opsoni-e# microorganisms0 an# the su*se8uent gro,th restriction or killing of
ingeste# microorganisms. $he antimicro*ial acti.ity of macrophages is me#iate# *y o1i#ati.e
an# non3o1i#ati.e mechanisms. $he o1i#ati.e antimicro*ial mechanisms0 ,hich inclu#e the
action of reacti.e o1ygen interme#iates 'nterleukin <B '!3<B" inhi*its interferon r.in#uce#
macrophage acti.ation for cytoto1icity against lar.ae of the human parasite Schistosoma
mansoni *y suppressing pro#uction of the to1ic effector molecule nitric o1i#e N/". 'n this
stu#y0 the mechanism of '!3<B action ,as i#entifie# as inhi*ition of en#ogenous tumor necrosis
factor a $NF3a" pro#uction *y interferon y3acti.ate# macrophages. $NF3a appears to ser.e as a
cofactor for interferon Ameate# acti.ation0 since *oth schismulum killing an# N/ pro#uction
,ere inhi*ite# *y anti3$NF3a anti*o#y0 ,hereas $NF3a alone ,as una*le to stimulate these
macrophage functions. '!3<B *locke# $NF3a pro#uction *y interferon .treate# macrophages
at the le.els of *oth protein an# mRN( synthesis. (##ition of e1ogenous $NF3a re.erse# '!3<B3
mel ate# suppression of macrophage cytoto1ic acti.ity as ,ell as N/ pro#uction. !ike,ise0
a##ition of a macrophage3triggering agent *acterial lipopolysacchari#e or muramyl #ipepti#e"0
,hich in#uce# the pro#uction of$NF3a0 also re.erse# the suppressi.e effect of '!3<B on
cytoto1ic function. 'n contrast to '!3<B0 t,o other cytokines0 '!3@ an# transforming gro,th
factor %0 ,hich also inhi*it macrophage acti.ation for shi n klg an# N/ pro#uction0 #i# not
su*stantially suppress en#ogenous $NF3a pro#uction. $hese results0 therefore0 #escri*e a
separate path,ay *y ,hich macrophage micro*ici#al function is inhi*ite# *y the #o,n3
regulatory cytokine '!3<B.
8uantitation o* mic$o%ici!a# acti'ity o* mononuc#ea$ 1agocyte"2 an in 'it$o tec1ni.ue3
(n in .itro assay techni8ue ,as set up to #etermine the phagocytic an# micro*ici#al acti.ity of a
monocyte3macrophage cell line using Can#i#a species as test organisms. $he norms ,ere
#etermine# for the acti.ity of peritoneal macrophages of rats I@.E> G+3 I.EZ phagocytosis an#
%L.@ G+3 L.IIZ 'CN" an# human I?.=> G+3 %.E%Z phagocytosis an# LB.>< G+3 E.%Z 'CN". $he
assay techni8ue ,as use# to test the #egree of acti.ation of macrophages in#uce# *y
metroni#a-ole0 $inospora cor#ifolia an# (spara8us racemousus an# to compare their effects
,ith a stan#ar# immunomo#ulator muramyl3#ipepti#e. (ll the three test agents increase# the
phagocytic an# killing capacity of macrophages in a #ose #epen#ent manner upto a certain #ose0
*eyon# ,hich either these acti.ities ,ere foun# to ha.e plateaue# or #ecrease#. $he optimal
#oses for MD:0 Metroni#a-ole0 (sparagus racemosus an# $inospora cor#ifolia ,ere foun# to *e
<BB micrograms0 %BB mg+kg0 IBB mg+kg an# <BB mg+kg respecti.ely. :atients ,ith cirrhosis
,ere screene# for #efects in monocyte function. $he #epresse# monocyte function IB.L= G+3
LZ phago an# @<.I@ G+3 <I.<>Z 'CN5 : M B.BL" ,as o*ser.e# in#icating a compromise# host
#efense. $he utility of this can#i#ici#al assay in e1perimental an# clinical stu#ies is #iscusse#.
In6'it$o e&e$imentation
Macrophages treate# ,ith culture flui#s from E!3@ cells0 a continuous $ cell line0 ,ere acti.ate#
to kill mNS(3$93L fi*rosarcoma cells0 amastigotes of !eishmania tropica0 an# schistosomula of
Schistosoma mansoni. (cti.e E!3@ factors elute# from Sepha#e1 ;3<BB in t,o #istinct regions:
molecular ,eight @L0BBB acti.ities in#uce# killing of unrelate# intracellular an# e1tracellular
targets" an# molecular ,eight I%0BBB acti.ities in#uce# killing of e1tracellular targets only".
$hese results confirm heterogeneity among acti.ation signals for the in#uction of macrophage
micro*ici#al an# tumorici#al acti.ities. Factors that in#uce# cytoci#al acti.ity against
e1tracellular tumor cells an# shistosomula ,ere #istinct from those that in#uce# #estruction of
intracellular amastigotes.
UNIT I7 IMMUNO+ATHOLOGY
Sam#ing an! $e"e$'ation o* ti""ue"
Carcasses or remnants of #ea# animals0 faeces an# other *iological material foun# in the ,il#
may *e .ery useful for o*taining #ata a*out ,il# populations ,ith little #istur*ance of li.e
animals or their ha*itat. 't can no longer *e regar#e# as ethical to kill threatene# ,il# animals
for o*taining skins for collections5 carcasses of animals foun# or confiscate# from poachers may
ser.e as a *etter source of material for reference collections. $herefore0 careful consi#eration
,hich parts of a specimen ha.e to *e #amage# or #estroye# for e1amination0 an# preser.ation of
,hich parts in reference collections is more useful one of us: (. Nekaris5 see also ;ro.es0 IBBI
in press"
Secimen" :1ic1 may %e u"e*u# in $e*e$ence co##ection"2
Skins5 stu#y skins0 mounte# specimens of the stu#y species the possi*ility of colour changes
cause# *y preser.ation shoul# *e consi#ere#"
Ket preser.e# specimens
Skeletal material0 skulls5 if no #ea# animals are a.aila*le an# killing of specimens is suppose# to
*e a.oi#e#0 for instance .ery #etaile# casts of #entition of anestheti-e# specimens ,ith #entistsk
materials are possi*le. /,l pellets may *e an interesting source of *ones one of us: C. ;ro.es"
2air samples5 reference hair collection of sympatric species

,o$ma#in $e"e$'ation2
(fter ,eighing an# measuring the animal an# attaching an a#e8uate la*el see la*elling"0 .ery
small specimens up to <BB g" can *e fi1e# ,hole *y su*merging them in <B Z *uffere#
formalin tissue 3 formalin solution ratio of at least < : <I". the *o#y ca.ity can *e fille# ,ith
formalin solution *y injection until it is turgi# an# firm5 some formalin may also *e injecte#
un#er the skin0 into the *o#y ca.ity0 larger muscles an# organs. 'f hypo#ermic nee#les are not
a.aila*le0 the *o#y ca.ity can *e opene# .entrally *y making a slit instea#0 allo,ing the
formalin to enter. Neeping the mouth open ,ith a piece of ,oo# or cotton may later allo,
e1amination of teeth. $hen the ,hole *o#y can *e immerse# in formalin0 in the posture in ,hich
it is suppose# to stay permanently *ecause it ,ill har#en. $he ratio of formalin to carcass must
*e at least <I to < to assure a goo# fi1ation. $issues can *e left in *uffere# neutrali-e# formalin
for se.eral months0 *ut formalin har#ens specimens5 therefore0 after fi1ation0 longterm storage
in alcohol may *e *etter. (fter preser.ation the carcass shoul# therefore *e ,ashe# in ,ater an#
transferre# into ethanol for permanent storage0 see *elo,: longterm li8ui# preser.ation
Nagorsen0 :eterson0 <>=B5 Munson0 IBBB5 Ra*ino,it- et al.0 IBBB".
E8uipment necessary: formalin0 *uffer0 ,ater0 scalpel an# + or hypo#ermic syringes0 material for
permanent la*els0 containers not metal containers unless they are aci#3proof line#0 *ecause
corrosion of the metal ,oul# #iscolour the specimen" Nagorsen0 :eterson <>=B".Formalin0
ho,e.er0 has some #isa#.antages5 for instance it #iscolours the fur0 after a longish immersion0
softens the *ones one of us: C. ;ro.es" an# pre.ents further e1amination for micro*iology.
+$e"e$'ation in a#co1o#2
(fter ,eighing0 a ,hole animal can *e preser.e# in a container of alcohol ?B3>BZ". Remo.al
of the intestine prior to storage of the animal in alcohol is recommen#e# Ra*ino,it- et al.0
IBBB".
+$e"e$'ation %y coo#ing o$ *$ee;ing2
Remo.al of the skin ,ith insulating fur *efore cooling or free-ing may help to cool the carcass
#o,n more 8uickly Schoon0 lecture manuscript".
Free-ing is not recommen#e# if histological e1amination is planne# Ko*eser an# Spraker0
<>=B".
La%o$ato$y $ea$ation o* "5in" ea$#ie$ !$ie! in t1e *ie#! as #escri*e# *y Do,ning <>@L":
<" Rela1ation of the #ry skin *y soaking it in luke,arm tap ,ater0 usually o.er night.
I" Brief ,ashing of the rela1e# skin ,ith soap an# ,ater.
%" Rinsing of the skin in a #egreasing agent such as .arsol or car*on3tetrachlori#e5 if greasy0 the
skins ,ere allo,e# to stan# in it for half an hour or so.
@" Drying of the skin in sa,#ust0 using compresse# air to assist the #rying an# to *lo, the
sa,#ust out of the hair.
Colour changes cause# *y this metho#0 particularly *y soaking0 see *elo,
+$o%#em o* co#ou$ c1ange" o* 1ai$ !u$ing $e"e$'ation o* *u$
2air colour of li.e animals may #iffer from the colour of preser.e# specimen for se.eral
reasons. Some #yes like plant juice may cause a re##ish hair colour0 algae may cause a greenish
hair colour in certain ar*oreal species0 for instance in the sloth Bradypus or a *right green #orsal
colour in )alagoides demidoff0 ,hich fa#es rapi#ly after the #eath of the animal San#erson
<>@B".
'n a##ition0 colour #ifferences in series of mammal skins may *e cause# *y preser.ation an#
storage metho#s rather than sho,ing the occurrence of #ifferent colour types re# an# grey
.arieties0 ran#om erythrism" in one species San#erson0 <>@B".
9se of c1emica#" may lea# to colour changes. Formalin #iscolours the fur one of us: C.
;ro.es". !ong immersion in solutions of alcohol0 salt0 alum or similar preser.ati.es also alters
colour Do,ning0 <>@L". Sumner <>I?" ,ho cleane# fur ,ith *en-ine or other chemical agents
for *etter compara*ility mentions colour changes.
4$ying met1o!" in the fiel# ha.e some influence. San#erson <>@B" foun# that series of skins
#rie# in *right sun in the fiel# ten#e# to turn re##ish. E1periments ,ith a maroon3coloure# rat0
Malacomys longipes0 sho,e# that furs *ecame grey0 #ark *ro,n or re##ish3*ro,n0 accor#ing to
,hether they ,ere #rie# in a close# container0 in sha#e or *right sunlight. Series of Praomys0
inclu#ing e1amples of *right re##ish an# oli.e3grey .arieties0 coul# *e e.enly #rie# to a
correspon#ing #ull siena ,hen su*mitte# to reciprocal treatment. Drying o.er a fire *y smoke
may also change colours San#erson <>@B".
Soa5ing o* !$ie! "5in" has a consi#era*le effect. Do,ning0 Cross an# :rince at the Royal
/ntario Museum of foology foun# a marke# #ichromatism in collectons of s8uirrel skins after
#ifferent preser.ation: ta,ny oli.e skins ha# *een ma#e up in the fiel#5 skins ,hich sho,e# a
#ark tone an# re##ish coloration ha# *een #rie# in the fiel# an# later rela1e# *y soaking in ,ater
an# ma#e up in the museum la*oratory. Some tests ,ith pieces of skin of a freshly kille# s8uirrel
sho,e# that treatment ,ith se.eral #ry preser.ati.es arsenic0 alum0 *ora10 salt" an# su*se8uent
#rying in an electric o.en at EBb Centigra#e for I@ hours #i# not lea# to colour changes0 *ut
soaking of further fresh an# #rie# samples0 ,ith an# ,ithout preser.ing chemicals a##e#0 in
,arm ,ater le# to color changes. (fter soaking originally ta,ny samples for one hour0 a *ora1
treate# an# salt treate# sample ha# *ecome ha-el0 an arsenic treate# sample *et,een ta,ny an#
russet0 an alum treate# sample *et,een russet an# ha-el0 an# e.en a #istille# ,ater treate#
sample sho,e# a percepti*le change. Samples that ha# *een soake# for longer perio#s sho,e#
further #arkening an# #eepening of color. (rsenic treate# an# #istille# ,ater treate# samples
sho,e# the least change0 *ora1 treate# the most change5 alum treate# an# salt treate# samples
,ere interme#iate. (lthough the a*o.e preser.ati.es increase# the amount of change ,hich took
place0 e.en soaking the skin in #istille# ,ater cause# a marke# alteration of the normal hair
color. E1amination of the museum collections of other species sho,e# similar changes after
soaking0 although less se.ere than in sciuri#ae0 *esi#es re##ening the yello, an# *uffy colors
ha# *ecome much #eeper in tone an# e1hi*ite# a cinnamon0 pink or re##ish cast. Changes ,ere
not e.enly #istri*ute# o.er the *o#y5 certain parts change# more than others. Do,ning
conclu#es that such changes in color may ren#er specimens almost useless for ta1onomic stu#ies
in ,hich color is an essential character0 that more a#e8uate metho#s for cleaning an# rela1ing
skins are necessary0 possi*ly ,ith minimi-ing of the time in ,hich the skin is moist0 an# that
such possi*ly colour3changing treatment must *e note# on la*els Do,ning0 <>@L".
!ater changes of colour of hair in museum specimens may occur #ue to fa#ing *ecause of
e1posal to light0 ol# age0 pro1imity of certain chemicals0 ra#iators an# other influences
San#erson <>@B".

Hai$"E 1ai$ $e*e$ence co##ection"
2air may ha.e microscopically .isi*le features allo,ing ta1onomic i#entification. 2air may not
only *e collecte# from li.e animals or carcasses0 *ut also for instance ,ith hair tu*es for
smaller mammals: tu*es of a ,i#th slightly larger than the stu#y species ,ith #ou*le3si#e#
sticky tape stuck to the insi#e" or hair catchers facilities ,ith ,ire3*rush3like structures5 animals
are encourage# to s8uee-e through"0 *aite# ore just attache# ,here animals are likely to pass0
left in the fiel# for <3I ,eeks.
Comparison ,ith ta1onomically i#entifie# hair samples of sympatric species either from an
o,n hair reference collection or in museum collections" may allo, i#entification of foo# + prey
of the stu#y species0 of remnants of the stu#y species in carni.ore faeces or o,l pellets or hairs
for instance collecte# from nests or ,ith hair catchers.
Hai$ "am#e" *o$ 4NA ana#y"i"
2air must *e plucke# not cut" to inclu#e follicle cells. ( minimum of <B3IB hairs shoul# *e
o*taine# (f( :rosimian $a1on (#.isory ;roup0 IBBI". Bear#er et al. <>>E" recommen# to
collect especially the long guar# hairs plucke# from *et,een the shoul#er *la#es an# hairs from
scent glan#s. !oose hairs ,hich can easily *e remo.e# *y plugging may alrea#y *e #ea# ,ith
little follicle cells ,ith DN( left one of us: Ch. Roos". $o pre.ent contamination from human
skin0 use of clean glo.es or an instrument for plugging is recommen#e#. Samples shoul# *e
store# in paper not plastic" en.elopes5 no special preser.ation is necessary (f( :rosimian
$a1on (#.isory ;roup0 IBBI".

$gg2 Ovarian Tissue2 $m&ryo2 and Sperm 3ree,ing
/ur cryopreser.ation sperm0 egg0 em*ryo0 an# o.arian tissue storage" program is one of the
*est in the ,orl#. Ke ha.e a LLZ pregnancy rate per cycle ,ith fro-en em*ryos0 ,hich is no
#ifferent than for fresh0 or unfro-en0 em*ryos. /ur fro-en em*ryo sur.i.al is almost >>Z.
$herefore0 if you ha.e 4e1tra4 .ia*le em*ryos resulting from your '&F proce#ure0 you can feel
comforta*le allo,ing us to free-e them.
Ke can no, also offer egg free-ing for ,omen ,ho ,ish to #elay chil#*earing0 an# ,ho ,ant to
preser.e their fertility for the future. (lthough the free-ing of embryos has *een 8uite relia*le0
the free-ing of eggs ,as for a long time only e1perimental0 an#0 until recently0 not .ery
successful. 2o,e.er0 the 'nfertility Center of St. !ouis has *egun a partnership ,ith the Nato
!a#ies Clinic in $okyo to *ring an e1citing ne, techni8ue0 0 to the 9nite# States. Kith this
metho#0 ,e can Fflash3free-eF unfertili-e# eggs ,ith a remarka*le effecti.eness0 an# then tha,
them at any later #ate for reimplantation. Ke can also offer o.arian tissue free-ing for ,omen
,ho #o not ,ant to un#ergo '&F in the future *ut ,ho just ,ant to concei.e naturally.
$he classic pro*lem ,ith free-ing eggs use# to *e that as one lo,ere# the temperature *elo, the
free-ing point0 the eggFs uncom*ine# genetic material rea#y for fertili-ation0 *ut also in a
comple1 an# #elicate state" ,oul# suffer #amage #ue to ice crystals forming insi#e the cell. 't
,as only possi*le to free-e em*ryos0 in ,hich the genetic material ha# alrea#y com*ine# ,ith
that from the sperm0 an# sta*ili-e#. $he classic free-ing techni8ues ,hich ha.e *een kno,n
since <>=%" ,ere *ase# on trying to e1tract ,ater from the cell as the temperature #rops0 to
minimi-e ice crystal #amage. $his has all change# no, ,ith the #e.elopment of our ne,
.itrification techni8ues. Ke no longer ha.e to play a tenuous game of minimi*ing ice crystal
formation0 ,e can no, entirely a!oid it 33 so that there is no internal #amage to the egg
,hatsoe.er.
Ke also offer .ery e1cellent 8uality o.arian tissue free-ing. $his proce#ure can *e use# to
preser.e the possi*ility of future fertility for ,omen ,ho are a*out to un#ergo ra#iation or
chemotherapy for cancer. 't may also *e use# for any ,omen ,ho may not *e a*le to plan for
chil#ren until they are ol#er. Ke ha.e ha# many successful o.arian tissue transplants an#
pregnancies0 an# so o.arian tissue free-ing has e1cellent promise for preser.ing your natural
fertility.
Sperm free-ing may *e nee#e# if the hus*an# is a*out to un#ergo ra#iation or chemotherapy for
cancer0 or is *eing #eploye# to a ,ar -one0 *ut ,ants to father chil#ren later. 'n a##ition0 sperm
may *e fro-en if the hus*an# has no sperm in his ejaculate an# re8uires a onetime surgical
sperm e1traction proce#ure0 or simply0 if the hus*an# is afrai# that on the #ay of your '&F
proce#ure he may *e too ner.ous to pro.i#e a specimen. (gain0 you can *e .ery confi#ent in the
relia*ility of our sperm free-ing an# storage system.
3ree,ing $ggs or $m&ryos &y the 4itrification Process
$his ne, techni8ue of free-ing calle# O.itrificationP a.oi#s the #amage cause# *y ice forming
insi#e the cell *y not trying to pull e.ery last molecule of ,ater out0 *ecause it is impossi*le to
#o this <BBZ. 'n fact0 ?BZ of the cell is ,ater0 an# at *est you can re#uce that to %BZ. So ,ith
the con.entional controlle# rate slo,3free-ing techni8ue0 there is al,ays going to *e some intra3
cellular ice crystal formation0 causing some #amage to em*ryos0 an# se.erely #amaging most
eggs. &itrification uses a super high concentration of antifree-e DMS/ an# ethylene glycol"0
an# #rops the temperature so rapi#ly that the ,ater insi#e the cell ne.er *ecomes ice. 't just
instantaneously super3cools into a soli# ,ith no ice crystal formation at all.
Ke can no, free-e an# tha,0 an# e.en refree-e an# retha,0 ,ith impunity0 using this ne,
protocol from Dr. Masashige Nu,ayama from the Nato Clinic in $okyo. Kith con.entional
Oslo, free-ing0P the temperature of the em*ryo goes #o,n at precisely B.%bC per minute. Kith
.itrification using four times the concentration of antifree-e0 or cryoprotectant"0 the temperature
is #roppe# at I%0BBB #egrees Cb per minute0 that is ?B0BBB times faster. (t that spee# of cooling0
an# at that concentration of antifree-e0 ice crystals simply cannot form.
/f course0 it is not 8uite as simple as it might soun#. Such high concentrations of antifree-e0 in a
fe, minutes0 coul# *e to1ic to cells. $herefore0 the em*ryos or eggs" must first *e place# in
lo,er concentrations of antifree-e an# sucrose to #ra, some ,ater out"0 an# then left in high
concentrations only for less than a minute *efore instantaneous free-ing. $hen ,hen the time
comes to tha, the em*ryo0 it must *e instantaneously ,arme#0 imme#iately taken out of the
high concentration of antifree-e0 an# then place# into a solution ,ith lo,er concentration0 in
or#er to a.oi# antifree-e to1icity. $his re8uires more skill than con.entional free-ing0 *ut it is
faster0 cheaper0 an# most importantly0 a.oi#s almost all free-ing #amage to either eggs or
em*ryos. Such a relia*le metho# of em*ryo free-ing gi.es the '&F program much greater a*ility
to a.oi# #angerous multiple pregnancy0 an# makes sche#uling for proce#ures like egg #onation
simpler for the patient.
9sing this .itrification techni8ue for free-ing0 ,e can relia*ly preser.e eggs as ,ell as em*ryos
so that the pregnancy rate is no #ifferent than if the eggs or em*ryos ha# ne.er *een fro-en. $his
allo,s us to preser.e the fertility of young ,omen for the future if they ,ish to #elay
chil#*earing0 *ut not lose their fertility as they age.
Ti""ue "am#e $e"e$'ation tec1ni.ue" in mic$og$a'ity an! $e#ate! im#ication" *o$
1a$!:a$e !e"ign3
Chemical fi1ation is an essential metho# of preser.ing *iological specimens #uring space flight
for #etaile# analyses after return to earth3*ase# la*oratories. 'n the #esign of the ;ra.itational
Biology Facility ;BF"0 it ,as #etermine# that a high percentage of ;BF e1periments in cell0
#e.elopmental an# plant *iology ,ill necessitate tissue preser.ation for a *roa# range of
specimen types. $ra#e3offs *et,een scientific re8uirements for sample preser.ation an#
har#,are #esigns are impacte# *y space station safety re8uirements an# logistics constraints. (
re.ie, of chemical fi1ati.es pre.iously flo,n0 an# #iscussion of other specimen preser.ation
techni8ues0 increases our un#erstan#ing of in3flight specimen fi1ation0 as ,ell as re.eals
strategies for impro.e# performance in this area. ;BF har#,are #esign0 cost0 an# sche#ule are
impacte# *y these issues.
I!enti*ication O* 7a$iou" Ce## Tye" An! Antigen" In Ti""ue"
'#entifying the antigens that ha.e the potential to trigger en#ogenous antitumor responses in an
in#i.i#ual cancer patient is likely to enhance the efficacy of cancer immunotherapy0 *ut current
metho#ologies #o not efficiently i#entify such antigens. $his stu#y #escri*es ,hat ,e *elie.e to
*e a ne, metho# of comprehensi.ely i#entifying can#i#ate tissue antigens that spontaneously
cause $ cell responses in #isease situations. Ke use# the ne,ly #e.elope# automate#0 t,o3
#imensional chromatography system :FID to fractionate the proteome of human tumor tissues
an# teste# protein fractions for recognition *y pree1isting tumor3specific CD@G $he cells an#
C$!s. (pplying this metho# using mice transgenic for a $CR that recogni-es an /&( pepti#e
presente# *y M2C class '0 ,e #emonstrate# efficient separation0 processing0 an# cross3
presentation to CD=G $ cells *y DCs of /&( e1presse# *y the /&(3transfecte# mouse
lymphoma RM(3/&(. (pplying this metho# to human tumor tissues0 ,e i#entifie# M9C< an#
E;FR as tumor3associate# antigens selecti.ely recogni-e# *y $ cells in patients ,ith hea# an#
neck cancer. Finally0 in an e1emplary patient ,ith a malignant *rain tumor0 ,e #etecte# CD@G
an# CD=G $ cell responses against t,o no.el antigens0 transthyretin an# calgranulin B+S<BB(>0
,hich ,ere e1presse# in tumor an# en#othelial cells. $he immunogenicity of these antigens ,as
confirme# in @ of <B other *rain tumor patients. $his fast an# ine1pensi.e metho# therefore
appears suita*le for i#entifying can#i#ate $ cell antigens in .arious #isease situations0 such as
autoimmune an# malignant #iseases0 ,ithout *eing restricte# to e1pression *y a certain cell type
or 2!( allele.
I!enti*ication o* ma0o$ ce## tye" in a$a**in "ection" o* %o'ine ti""ue"
A%"t$act
'#entification of cell types in *o.ine tissue sections is complicate# *y the limite# a.aila*ility of
anti3*o.ine anti*o#ies0 an# *y antigen retrie.al treatments re8uire# for formalin3fi1e# tissue
samples. Ke ha.e e.aluate# an anti*o#y an# lectin panel for i#entifying major cell types in
paraffin3em*e##e# *o.ine tissue sections0 an# report optimi-e# pretreatments for these markers.
$he panel of markers allo,s the i#entification of all major cell types in paraffin3em*e##e# cattle
tissue sections *y immunohistochemistry or lectin histochemistry. 2eat3in#uce# epitope retrie.al
metho#s are re8uire# for most anti*o#ies.
Int$o!uction
Specific i#entification of cell types in *o.ine tissues is hin#ere# *y the limite# a.aila*ility of
anti3*o.ine anti*o#ies. $he species cross3reacti.ity information of other commercially a.aila*le
anti*o#ies is also often limite#. $hus0 suita*le anti*o#ies must *e searche# for *y trial an# error.
$his is further complicate# *y the fact that for many anti*o#ies a successful immunostaining is
on#y accom#i"1e! a*te$ an otimi;e! antigen $et$ie'a# t$eatment3
Met1o!
$issue samples ,ere o*taine# from slaughtere# animals. $he use of animals ,as appro.e# *y
the animal ethics committee of the 9ni.ersity of 2elsinki. $issue samples ,ere fi1e# either in
@Z phosphate3*uffere# paraformal#ehy#e :F(" for I@ hours at G@bC or in <BB Z ethanol for I
hours at G@bC follo,e# *y <IB hours at 3IBbC0 em*e##e# in paraffin0 an# sectione# to IA@ am
:F(" or @ am ethanol" sections.
(nti*o#ies an# lectins
Immuno1i"toc1emi"t$y ,as performe# using either the (BC metho# a.i#in *iotin comple1"
or tyrami#e amplification0 using Shan#on Co.erplates $hermoElectron". :araffin3em*e##e#
sections ,ere #e,a1e#0 rehy#rate#0 su*jecte# to an antigen retrie.al proce#ure see *elo,"0 an#
permea*ili-e# ,ith B.<Z to <Z $,een3IB in phosphate3*uffere# saline :BS". $he sections
,ere then *locke# for en#ogenous *iotin0 ,ith <BZ egg ,hite po,#er in ,ater as an a.i#in
solution" an# < mg+ml D3*iotin Sigma3(l#rich0 St. !ouis0 M/" in :BS0 ,hen necessary0 an# for
nonspecific *in#ing ,ith <Z goat serum in :BS. $hey ,ere incu*ate# in the primary anti*o#y
o.ernight at G@bC0 in :BS containing <Z *o.ine serum al*umin0 ,ashe#0 an# incu*ate# ,ith
goat *iotinylate# anti3mouse or anti3ra**it secon#ary anti*o#y Dako0 ;lostrup0 Denmark" for I
hours in room temperature. $he (BC #etection ,as performe# using the &ectastain Elite (BC
kit an# the D(B #iamino*en-i#ine" su*strate kit *oth &ector !a*oratories0 Burlingame0 C("
accor#ing to manufacturerFs instructions. For tyrami#e amplification0 sections ,ere incu*ate# in
a.i#in D 3conjugate# pero1i#ase &ector"0 in *iotinylate# tyrami#e again in a.i#in3pero1i#ase0
an# in the D(B su*strate. $he amplification typically allo,s four to ten times more #ilute
anti*o#y solutions than ,ith the (BC metho#. (ll sections ,ere counterstaine# ,ith MayerFs
hemato1ylin an# em*e##e# ,ith Faramount Dako".
Antigen $et$ie'a#
2eat3in#uce# antigen retrie.al ,as performe# in a stan#ar# kitchen micro,a.e o.en. $he sli#es
,ere heate# in LBB ml of retrie.al solution at ?LB K po,er for <L minutes for the caspase
anti*o#y0 <B minutes"0 follo,e# *y a cooling perio# of IB minutes for the caspase anti*o#y0 %B
minutes". $he follo,ing solutions ,ere use#: for aci# retrie.al0 LB mM glycine32Cl p2 %5 for
neutral retrie.al0 I l SSC p2 E so#ium chlori#e0 so#ium citrate *uffer"5 an# for alkaline
retrie.al0 <B mM $ris32Cl p2 >.L0 < mM ED$(.
:rotease3in#uce# antigen retrie.al ,as performe# in Co.erplates0 at %?bC for %B minutes0 ,ith
<B to LB ag+ml ethanol3fi1e# samples" or LB to <BB ag+ml :F(3fi1e# samples" protease :E><<
Sigma3(#rich" in <B mM $ris32Cl p2 ?.@0 B.L mM ED$(.
Mic$o"coy an! 1otog$a1y
$he staine# sections ,ere .ie,e# ,ith a !eica DM@BBB microscope an# photographe# using a
S'S Color.ie, <I #igital camera.
4i"cu""ion
(s a *y3pro#uct of a research project on stem cell fates0 ,e ha.e e.aluate# a selection of
anti*o#ies for i#entifying major *o.ine cell types in paraffin3em*e##e# tissue sections. Some of
these ha.e *een raise# against *o.ine antigens0 some are pre.iously kno,n to *e *o.ine cross3
reacti.e0 an# others ,e ha.e teste# ,ithout such prior kno,le#ge. /ptimal antigen retrie.al
metho#s for each anti*o#y are reporte#. 'n a##ition to anti*o#ies0 t,o lectins are presente#. $he
emphasis is on paraformal#ehy#e3fi1e# tissues0 *ut as some anti*o#ies are incompati*le ,ith
such material0 ,e ha.e also use# ethanol fi1ation.
Eit1e#ium
Se.eral anti3keratin anti*o#ies ,ere e.aluate# for epithelial markers. $he (E<+(E% monoclonal
anti*o#y cocktail0 raise# against human epi#ermal keratin0 ,as the most useful pan3epithelial
marker. Kith alkaline antigen retrie.al see Metho#s"0 most types of epithelia ,ere strongly an#
specifically staine# ,ith this anti*o#y. Neutral or protease3in#uce# retrie.al ,as sufficient for
some *ut not all tissues.
(nother pan3keratin anti*o#y !u3Lalso staine# most epithelia ,ith protease3in#uce# retrie.al0
*ut #i# not co.er all epithelia as comprehensi.ely as (E<+(E% in our han#s.
$he polyclonal pan3keratin anti*o#y teste# yiel#e# nonspecific staining of cells. $he high
molecular ,eight cytokeratin anti*o#y %@m E<pro.i#e# .ery strong staining of some epithelia
nota*ly epi#ermis an# li.er"0 *ut is o*.iously not as comprehensi.e as the pan3keratin markers.
Fe, *o.ine en#othelial markers are a.aila*le. $he anti3 .on Kille*ran# anti*o#yteste# stains
many *ut not all en#othelia. Nota*ly0 ne, *loo# .essels in granulation tissue ,ere strongly
staine#. $he lectins M!3' an# especially ;S! '3B@yiel#e# strong staining of en#othelial cells0
*ut they also stain some leukocyte populations. Ke faile# to o*tain a goo# staining ,ith the anti3
type '& collagen anti*o#y M%F?or ,ith the polyclonal anti3 en#othelial nitric o1i#e synthase
anti*o#y.
Connecti'e ti""ue
&imentin is a general marker for cells of the mesenchymal lineage. $he monoclonal anti3
porcine .imentin anti*o#y &> pro.i#e# strong an# specific staining e.en ,ithout antigen
retrie.al.
$he type ' procollagen anti*o#y S:<.D= staine# acti.e fi*ro*lasts in .arious connecti.e tissues.
$he *est staining ,as o*taine# ,ith the alkaline retrie.al metho#. Some nonspecific staining
,as seen0 in se*aceous glan#s for e1ample.
Mu"c#e
$he #esmin anti*o#y D%% staine# all muscle types0 performing *est after neutral antigen
retrie.al . $he muscle actin anti*o#y 22F%L recogni-es the alpha an# gamma isotypes present
in all muscle types. $he aci# an# alkaline retrie.al metho#s yiel#e# optimal results. $he [3
smooth muscle actin anti*o#y <(@ stains only a su*set of smooth muscle tissues0 #ue to the
more restricte# e1pression pattern of the antigen. $he circular muscle layer in the intestine ,as
not staine#. $he neutral antigen retrie.al metho# pro#uce# the *est results ,ith this anti*o#y. (ll
these anti*o#ies ,ere specific for muscle tissues.
Ne$'ou" ti""ue
$he monoclonal anti3 NeuN anti*o#y (EB ,as the most comprehensi.e neuronal marker teste#.
't staine# most neurons0 :urkinje cells *eing a nota*le e1ception. Best results ,ere o*taine#
,ith alkaline retrie.al.
$u*ulin an# neurofilament anti*o#ies are also useful as general neuronal markers. Majority of
neurons ,ere staine# *y *oth tu*ulin m''' anti*o#ies an# the pan3neurofilament cocktail0 an#
more restricte# su*groups *y the neurofilament <EB+IBB kD anti*o#y0 as e1pecte#.
(stroglia ,ere *eautifully staine# ,ith the polyclonal anti3;F(: anti*o#y0 ,ith most
pretreatments. /ligo#en#roglia ,ere successfully staine# ,ith the anti3CN:ase anti*o#y <<3LB0
,hereas the anti3/@ anti*o#y =<faile# to pro#uce any specific staining in our han#s. S<BBis a
more general marker for glial cells. 't is also e1presse# in se.eral cell types outsi#e the ner.ous
system.
$he microglial cells ,ere .ery ,eakly staine# ,ith the pan3leukocyte an# macrophage markers
teste#. $he mistletoe lectin M!3'yiel#e# successful staining ,ith all pre3treatments. 't also stains
most en#othelial cells.
Leu5ocyte"
!eukocyte markers are often species3specific0 an# most commonly use# in flo, cytometry.
$hus0 most of the anti*o#ies teste# ,ere raise# against *o.ine antigens0 *ut information on
histological applications ,as limite#.
/f the pan3leukocyte markers0 the anti3 CD@L anti*o#y C(C$BL<(0 ,orke# ,ell on ethanol3
fi1e# material ,ith mil# protease treatment0 e1cept for the microglia0 ,hich ,ere ,eakly
staine#. $he 8uality of the CC< anti*o#yappeare# to suffer from a change in the pro#uction
metho# #uring the research project.
Ke teste# t,o CD<<a+<= anti*o#ies0 of ,hich B($?L(,as useful ,ith aci# antigen retrie.al0
,hile M9C?E(faile# ,ith any pre3treatment.
For lymphocytes0 the CD%n $ cells" an# CD?>[ B cells" anti*o#ies raise# against synthetic
cytoplasmic pepti#es ,orke# ,ell ,ith antigen retrie.al0 pro.i#ing a strong an# specific
staining. $he anti3 immunoglo*ulin anti*o#ies successfully staine# B cells ,ith alkaline
retrie.al.
Myeloi# cells ,ere staine# ,ith the anti3 CD<<* anti*o#y MM<B(using aci# retrie.al or
ethanol3fi1e# material. $he macrophage marker CDE=,as also useful ,ith protease3treate#
sections0 *ut #i# not stain microglia. $he CD<@ anti*o#y MME<(,as only useful ,ith ethanol3
fi1e# material. $he polyclonal anti3lyso-yme anti*o#ystaine# a num*er of cells in the intestinal
epithelium0 for e1ample0 *ut ,e faile# to confirm the specificity of the staining.
Ce## "tatu"
$he proliferation marker M'B3<yiel#e# goo# staining ,ith alkaline antigen retrie.al. (poptotic
cells ,ere successfully staine# ,ith the clea.e# caspase % anti*o#yusing neutral retrie.al0
although the staining ,as not .ery strong.
Ta%#e ?
E.aluation of markers. GG: goo# staining strong0 specific"0 G: poor staining ,eak or inclu#ing
non3specifically staine# cells"0 3: unsuccesful staining. :F(: paraformal#ehy#e fi1ation0 Et/2:
ethanol fi1ation. N: no epitope retrie.al0 :: protease3in#uce# epitope retrie.al0 2%: heat3in#uce#
epitope retrie.al 2'ER" at p2 %0 2E: 2'ER at p2 E0 2>: 2'ER at p2 >.L. Metho#s: ( C a.i#in3
*iotin3comple10 $ C tyrami#e signal amplification. NF C neurofilament. Sources: D2SB C
De.elopmental Stu#ies 2y*ri#oma Bank0 BD C BD Biosciences0 N+! C NeoMarkers+!a*&ision0
&ector C &ector !a*oratories0 K+2 C Kitten+2er#ecke 9ni.ersity0 Ch C Chemicon0 SM C
Stern*erger Monoclonals0 *+C C *(*co+Co.ance0 &MRD: &eterinary Me#icine Research
Diagnostics0 SC C Santa Cru-0 B! C Bethyl !a*oratories0 Bm C Biome#a0 CS$ C Cell Signaling
$echnology.
Ma$5e$ c#one>tye Immunogen Sou$ce -e*3 Staining 4i#ution (met1o!)
+,A EtOH
N + H@ H6 HF N +
E:'$2E!'9M0 END/$2E!'9M
collagen$ype '& M%F?
human
collagen
DS2B 6<B7 3 3 3 3 3 3 3 <:IBB$
cytokeratin0
2MK
%@*etaE<I
human epi#.
keratin
Dako 6L7 GG 3 GG <:<BBB$
cytokeratin0 pan
ra**it
polycl.
*o.ine epi#.
keratin
fyme# n.a. G G G G <:<BBB$
eNos+N/S$ype
'''
ra**it
polycl.
pepti#e
human"
BD 6<<7 G 3 G G <:<BBB$
keratin0 pan (E< G (E%
human epi#.
keratin
N+! 6I0%7 3 GG 3 GG GG GG <:<BB(0 <:<BBB$
keratin0 pan !u3L
lung cancer
cell line
N+! 6@7 GG GG 3 GG <:<BB(0 <:<BBB$
lectin ;S! '3B@
).
simplicifolia
n.a. &ector 6=0>7 GG GG GG GG <:@BBB$
lectin M!3'
#iscum
album
n.a. K+2 6?7 GG GG GG GG GG <:IBBB$
.on Kille*ran#
ra**it
polycl.
human .KF Dako 6E7 G GG <:@BB(0 <:=BB$
C/NNEC$'&E $'SS9E
procollagen$ype
'
S:<.D=
o.ine
aminopropept.
DS2B 6<@7 G G G G GG 3 3 <:IBBB$
.imentin &>
porcine
.imentin
Dako 6<I0<%7 GG GG GG <:<BB(0 <:LBB$
M9SC!E
actin0 muscle 22F%L n.a. En-o 6<?7 G GG G GG GG GG <:<BB(
actin0 smooth
muscle [
<(@ pepti#e Dako 6<=7 GG G <:LBB$
#esmin D%% human #esmin Dako 6<L0<E7 G GG GG <:@BB(0 <:IBBB$
NE9R/N(! $'SS9E
CN:ase <<3LB
human *rain
CN:ase
Ch n.a. GG 3 GG 3 3 <:@BB(
;F(:
ra**it
polycl.
human ;F(: fyme# n.a. GG GG G GG GG GG <:IBB(
NeuN (EB
mouse
neuronal
nuclei
Ch 6<>0II7 G G 3 G GG GG G <:@BBB$
NF <EB+IBB kD RM#/3IB
rat
neurofilaments
fyme# 6II7 GG 3 3 GG GG <:@BB(
NF0 pan SM'%<< n.a. SM 6I<7 GG G GG GG G <:<BBB(0 <:IBBB$
Ma$5e$ c#one>tye Immunogen Sou$ce -e*3 Staining 4i#ution (met1o!)
+,A EtOH
N + H@ H6 HF N +
cockt"
/@ =< *o.ine *rain Roche 6I%7 3 3 3 3 <:I(
S<BB
ra**it
polycl.
*o.ine S<BB Dako 6I@7 GG <:=BB(
tu*ulin m'''
ra**it
polycl.
pepti#e rat" *+C n.a. 3 GG 3 G <:EBBB(0<:<BBBB$
tu*ulin m''' $93IB n.a. Ch 6IB7 GG GG GG GG GG <:@BB(0 <:%BBB$
!E9N/CJ$ES
CD<<a+<= M9C?E(
sheep0 pig
leukocytes
&MRD 6I=7 3 3 3 3 <:@B(
CD<<a+<= B($?L(
ruminant
leukocytes
&MRD 6I?7 3 GG 3 3 G <:@B(0 <:IBB$
CD<<* MM<B( n.a. &MRD 6%I7 GG GG <:LBB$
CD<@ MME<(
*o.ine
mononucl.
cells
&MRD 6IL7 G G <:IBB
CD%@
ra**it
polycl.
pepti#e
human"
SC n.a. G 3 3 <:IBB(0 <:IBB$
CD%n
ra**it
polycl.
pepti#e
human"
Dako 6I>7 3 GG G GG <:<BB(0 <:<BBB$
CD@L C(C$BL<(
*o.ine act.
lymphocytes
&MRD 6IL7 G G G G G GG <:=BB$
CD@L CC< n.a. Serotec 6IE7 G G <:<BB(
CDE= EBM<<
human
macrophages
Dako 6%%7 3 G 3 GG <:=B(
CD?>[cy 2ML?
pepti#e
human"
Dako 6%B7 3 G GG <:LBB$
'g(
ra**it
polycl.
*o.ine 'g( B! n.a. GG GG GG <:<BB(
'gM
ra**it
polycl.
*o.ine 'gM B! n.a. G GG <:<LBB(
'gM B'g?%( *o.ine 'g &MRD 6%<7 3 G GG <:LBBB(
lyso-yme
ra**it
polycl.
human
lyso-yme
Bm 6%@7 G 3 3 G G G <:LBB$
CE!! S$($9S
clea.e# caspase
% (sp<?L"
ra**it
polycl.
pepti#e
human"
CS$ 6%?7 GG G <:?L(
NiE? antigen M'B3<
pepti#e
human"
Dako 6%L0%E7
I"o#ation An! C1a$acte$i;ation O* Ce## Tye" ,$om In*#ammato$y Site" An! In*ecte!
Ti""ue"
Cellular component
$he cellular component in.ol.es leukocytes0 ,hich normally resi#e in *loo# an# must mo.e
into the inflame# tissue .ia e'tra!asation to ai# in inflammation. Some act as phagocytes0
ingesting *acteria0 .iruses0 an# cellular #e*ris. /thers release en-ymatic granules ,hich #amage
pathogenic in.a#ers. !eukocytes also release inflammatory me#iators ,hich #e.elop an#
maintain the inflammatory response. ;enerally speaking0 acute inflammation is me#iate# *y
granulocytes0 ,hile chronic inflammation is me#iate# *y mononuclear cells such as monocytes
an# lymphocytes.
$he process of acute inflammation is initiate# *y cells alrea#y present in all tissues0 mainly
resi#ent macrophages0 #en#ritic cells0 histiocytes0 Nupffer cells an# mastocytes.
Comparison *et,een acute an# chronic inflammation:
Acute C1$onic
:athogens0 injure# tissues
:ersistent acute inflammation #ue to non3#egra#a*le
pathogens0 persistent foreign *o#ies0 or autoimmune
reactions
Neutrophils0 mononuclear cells
monocytes0 macrophages"
Mononuclear cells monocytes0 macrophages0 lymphocytes0
plasma cells"0 fi*ro*lasts
&asoacti.e amines0 eicosanoi#s
'FN3^ an# other cytokines0 gro,th factors0 reacti.e o1ygen
species0 hy#rolytic en-ymes
'mme#iate Delaye#
Fe, #ays 9p to many months0 or years
Resolution0 a*scess formation0
chronic inflammation
$issue #estruction0 fi*rosis
?3 T1e i"o#ation an! c1a$acte$i;ation o* g#o%u#e #eucocyte"2 t1ei$ !e$i'ation *$om muco"a#
ma"t ce##" in a$a"iti;e! "1ee
/.ine mucosal mast cells an# glo*ule leucocytes ha.e *een isolate# from the a*omasum of
normal sheep0 an# from animals challenge# ,ith /stertagia circumcincta. $he ultrastructural0
morphological an# histochemical properties of these cells ha.e *een in.estigate#. $he granules
of o.ine mucosal immunoglo*ulin an# a serine esterase. $hese cells also possess surface
immunoglo*ulin. Cells morphologically interme#iate *et,een mucosal mast cells an# *ulin.
Cells morphologically interme#iate *et,een mucosal mast cells an# glo*ule leucocytes ha.e
similar granule an# surface properties. $hese o*ser.ations0 together ,ith 8uantitati.e #ata0
in#icate that alterations in the granule structure of mucosal mast cells as a conse8uence of
prolonge# antigenic challenge gi.e rise to mast cells in the epithelium ,hich0 in the past0 ha.e
*een commonly recogni-e# as glo*ule leucocytes.
23 I"o#ation an! c1a$acte$i;ation o* g$anu#ocyte #y"o"oma# $otein" an! "tu!y o* t1ei$
e**ect" on t1e c#otting "y"tem3
!ysosomes granules" of ra**it :MN leukocytes ,ere e1tracte# ,ith either 2Cl or 2IS/@0 an#
the e1tracts ,ere chromatographe# o.er Sepha#e1 to separate protein constituents. Some of the
lo, molecular ,eight cationic proteins homogeneous on SDS :(;E =Z an# <I.LZ gels" ,ere
characteri-e# *y electrophoretic mo*ility in aci# gels an# *y amino aci# analysis. ( %0?BB #alton
polypepti#e0 rich in arginine an# cysteine0 prolonge# the partial throm*oplastin time of normal
plasma. 'n lo, concentration0 this protein shortene# the clotting time of pure fi*rinogen *y
throm*in. 'n high concentration this lysosomal cationic protein precipitate# fi*rinogen from
solution5 no fi*rinopepti#es ,ere release# to suggest clea.age of fi*rinogen. Fi*rinolytic
protease acti.ity ,as #etecte# in cru#e 2IS/@ e1tracts *ut not in cru#e 2Cl e1tracts. $,o
separate plasminogen acti.ators0 #iffering from kallikrein or prekallikrein0 ,ere isolate# from
the 2IS/@ lysosomal e1tract an# ,ere partially characteri-e#5 neither e1hi*ite# proteolytic
acti.ity on fi*rinogen free of plasminogen.
@3 I"o#ation an! c1a$acte$i;ation o* LMCE a no'e# #ym1ocyte an! monocyte
c1emoatt$actant 1uman CC c1emo5ineE :it1 mye#o"u$e""i'e acti'ity3
By searching the E1presse# Se8uence $ag ES$" #ata *ase0 ,e i#entifie# a partial cDN(
se8uence enco#ing a no.el human CC chemokine. $he entire cDN( se8uence ,as #etermine#
an# re.eale# a CC chemokine ,hose mature protein consiste# of <BB amino aci#s ,ith pre#icte#
molecular ,eight of << k#. $he chemokine preferantially chemoattracte# lymphocytes an#
monocytes *ut not neutrophils. 't ,as0 therefore0 name# !MC !ymphocyte an# Monocyte
Chemoattractant". !MC e1hi*ite# potent myelosuppressi.e acti.ity0 ,hich ,as compara*le to
that of M':3<alpha. Ke i#entifie# se.eral *acterial artificial clones B(C" containing the !MC
gene along ,ith t,o human CC chemokine su*family mem*ers5 leukotactin3< !kn3<" an#
CN*eta=3<+CN*eta=. $his #ata suggests that the !MC gene is locate# at human chromosome
<?8 ,hich encompasses a human CC chemokine gene cluster.
43 I"o#ation an! c1a$acte$i;ation o* mac$o1age" *$om $at em%$yonic mu"c#e cu#tu$e.
Ke ha.e pre.iously #escri*e# the ,i#e #istri*ution of resi#ent macrophages in normal rat
skeletal muscle. 'n this stu#y0 ,e in.estigate# the characteristics of the macrophages that occur
in rat em*ryo muscle cultures. Ke sho,e# that cells of monocyte3macrophage lineage are
present in primary muscle cultures of rat em*ryos <= #ays in gestation" an# that these cells
form morphologically an# phenotypically heterogeneous populations0 *ase# on their reaction
,ith monoclonal anti*o#ies ED<0 EDI0 ED%0 an# /X@%. Constituti.ely 'aG cells ,ith #en#ritic
appearance ,ere also o*ser.e#. Furthermore0 ,e esta*lishe# the proce#ure for isolation of
macrophages from the primary muscle cultures. $he isolate# cells0 mostly ED<G0 e1presse#
class ' an# CD@ antigens an# *ore complement C%" receptors on their surfaces. $he fact that
cell of monocyte3macrophage lineage occur in the em*ryonic muscle suggests that #uring
em*ryogenesis these cells may enter the #e.eloping muscle an# gi.e rise to a population of
tissue3associate# macrophages.
B3 I"o#ation an! c1a$acte$i;ation o* !en!$itic ce##" *$om common ma$mo"et" *o$ $ec#inica#
ce## t1e$ay "tu!ie"
Den#ritic cells DCs" ha.e important functions as mo#ulators of immune responses0 an# their
a*ility to acti.ate $ cells is of great .alue in cancer immunotherapy. $he isolation of DCs from
the peripheral *loo# of rhesus an# (frican green monkeys has *een reporte#0 *ut the immune
system in the common marmoset remains poorly characteri-e#0 although it offers many potential
a#.antages for preclinical stu#ies. 'n the present stu#y0 ,e #e.ise# metho#s0 *ase# on
techni8ues #e.elope# for mouse an# human DC preparation0 for isolating DCs from three major
tissue sources in the common marmoset: *one marro, BM"0 spleen an# peripheral *loo#. Each
set of separate# cells ,as analyse# using the cell surface DC3associate# markers CD<<c0 CD=B0
CD=%0 CD=E an# human leucocyte antigen 2!("3DR0 all of ,hich are anti*o#ies against
human antigens0 an# the cells ,ere further characteri-e# *oth functionally an# morphologically
as antigen3presenting cells. BM pro.e# to *e an e1cellent cell source for the isolation of DCs
inten#e# for preclinical stu#ies on cell therapy0 for ,hich large 8uantities of cells are re8uire#. 'n
the BM3#eri.e# CD<<c
G
cell population0 cells e1hi*iting the characteristic features of DCs ,ere
enriche#0 ,ith the typical DC morphology an# the a*ilities to un#ergo en#ocytosis0 to secrete
interleukin '!"3<I0 an# to stimulate Xenogenic $ cells. Moreo.er0 BM3#eri.e# DCs pro#uce#
the neurotrophic factor N$3%0 ,hich is also foun# in murine splenic DCs. $hese results suggest
that BM3#eri.e# DCs from the common marmoset may *e useful for *iological analysis an# for
preclinical stu#ies on cell therapy for central ner.ous system #iseases an# cancer.
63 I"o#ation an! c1a$acte$i"ation o* a ma"t ce## !eg$anu#ating "u%"tance *$om Ascaris suum
<. During in.estigations of allergic phenomena associate# ,ith parasitic infestations0 a su*stance
,hich in#uces #egranulation of mast cells has *een isolate# from the *o#y flui# of +scaris
suum. 't has *een assaye# *y its a*ility to cause #egranulation of rat peritoneal mast cells in
!itro.
I. $he mast cell factor has reasona*le sta*ility to heat. $o retain mast cell factor acti.ity #uring
isolation an# storage of the .arious preparations0 it ,as essential to pre.ent aggregation of the
mast cell factor0 presuma*ly resulting from o1i#ation of sulphy#ryl groups. (ggregation ,as
pre.ente# *y incorporation in the *uffers of I3mercaptoethanol an# #ithiothreitol. :recautions
,ere also necessary to a.oi# loss of acti.ity #uring concentration of fractions particularly
*ecause the molecular si-e of mast cell factor *arely permitte# its retention *y the usual #ialysis
mem*ranes.
%. $he scheme a#opte# for purification of mast cell factor in.ol.e# successi.e chromatographic
fractionations on DE(E3cellulose0 Sepha#e1 ;3?L an# SE3Sepha#e1. $he pro#uct ga.e a single
*an# on polyacrylami#e3gel electrophoresis in gra#ient an# ?Z gels0 as ,ell as in gels
containing so#ium #o#ecyl sulphate. 'n the last system proteins ,ere first re#uce# an# unfol#e#5
*y comparison in #o#ecyl sulfate gels of the mo*ility of mast cell factor ,ith that of reference
proteins0 mast cell factor ,as estimate# to ha.e a molecular ,eight of ==BB.
@. $he se#imentation3.elocity pattern sho,e# a single peak ,ith an s .alue of <.B?0 a result
correspon#ing to a molecular ,eight of the or#er of =EBB. (mino aci# analysis in#icate# a
molecular ,eight of =>%?. $he a*o.e e.i#ence collecti.ely suggests that the isolate# preparation
of mast cell factor is su*stantially homogeneous.
C3 I"o#ation an! C1a$acte$i;ation o* a No'e# In!uci%#e Mamma#ian Ga#ectinK
( no.el mammalian galectin cDN( o!gal,," ,as isolate# *y representational #ifference
analysis from sheep stomach a*omasal" tissue infecte# ,ith the nemato#e parasite0
&aemonchus contortus. $he mRN( is greatly up3regulate# in helminth lar.al infecte#
gastrointestinal
tissue su*ject to inflammation an# eosinophil infiltration. 'mmunohistological analysis in#icates
that the protein is locali-e# in the cytoplasm an# nucleus of upper epithelial cells of the
gastrointestinal tract. $he protein is also #etecte# in mucus samples collecte# from infecte#
a*omasum *ut not from uninfecte# tissue. $he restricte# an# in#uci*le e1pression of o!gal,,
mRN( an# limite# secretion of the protein support the hypothesis that /&;(!<< may *e
in.ol.e# in gastrointestinal immune+inflammatory responses an# possi*ly protection
against infection.
,unctiona# Stu!ie" On I"o#ate! Ce##"
?3 Immunocytoc1emi"t$y
'mmunocytochemistry la*els in#i.i#ual proteins ,ithin cells0 such as $2 green" in the a1ons of
sympathetic autonomic neurons.
Immunocytoc1emi"t$y ICC" is a common la*oratory techni8ue that uses anti*o#ies that target
specific pepti#es or protein antigens in the cell .ia specific epitopes. $hese *oun# anti*o#ies can
then *e #etecte# using se.eral #ifferent metho#s. 'CC allo,s researchers to e.aluate ,hether or
not cells in a particular sample e1press the antigen in 8uestion. 'n cases ,here an
immunopositi.e signal is foun#0 'CC also allo,s researchers to #etermine ,hich su*3cellular
compartments are e1pressing the antigen.
5mmunocytochemistry vs* immunohistochemistry
'mmunocytochemistry #iffers from immunohistochemistry in that the former is performe# on
samples of intact cells that ha.e ha# most0 if not all0 of their surroun#ing e1tracellular matri1
remo.e#. $his inclu#es cells gro,n ,ithin a culture0 #eposite# from suspension0 or taken from a
smear. 'n contrast0 immunohistochemical samples are sections of *iological tissue0 ,here each
cell is surroun#e# *y tissue architecture an# other cells normally foun# in the intact tissue.
'mmunocytochemistry is a techni8ue use# to assess the presence of a specific protein or antigen
in cells culture# cells0 cell suspensions" *y use of a specific anti*o#y0 ,hich *in#s to it0 there*y
allo,ing .isuali-ation an# e1amination un#er a microscope. 't is a .alua*le tool for the
#etermination of cellular contents from in#i.i#ual cells. Samples that can *e analy-e# inclu#e
*loo# smears0 aspirates0 s,a*s0 culture# cells0 an# cell suspensions.
$here are many ,ays to prepare cell samples for immunocytochemical analysis. Each metho#
has its o,n strengths an# uni8ue characteristics so the right metho# can *e chosen for the
#esire# sample an# outcome.
Cells to *e staine# can *e attache# to a soli# support to allo, easy han#ling in su*se8uent
proce#ures. $his can *e achie.e# *y se.eral metho#s: a#herent cells may *e gro,n on
microscope sli#es0 co.erslips0 or an optically suita*le plastic support. Suspension cells can *e
centrifuge# onto glass sli#es cytospin"0 *oun# to soli# support using chemical linkers0 or in
some cases han#le# in suspension.
Concentrate# cellular suspensions that e1ist in a lo,3.iscosity me#ium make goo# can#i#ates
for smear preparations. Dilute cell suspensions e1isting in a #ilute me#ium are *est suite# for the
preparation of cytospins through cytocentrifugation. Cell suspensions that e1ist in a high3
.iscosity me#ium0 are *est suite# to *e teste# as s,a* preparations. $he constant among these
preparations is that the ,hole cell is present on the sli#e surface. For any intercellular reaction to
take place0 immunoglo*ulin must first tra.erse the cell mem*rane that is intact in these
preparations. Reactions taking place in the nucleus can *e more #ifficult0 an# the e1tracellular
flui#s can create uni8ue o*stacles in the performance of immunocytochemistry. 'n this situation0
permea*ili-ing cells using #etergent $riton X3<BB or $,een3IB" or choosing organic fi1ati.es
acetone0 methanol0 or ethanol" *ecomes necessary.
(nti*o#ies are an important tool for #emonstrating *oth the presence an# the su*cellular
locali-ation of an antigen. Cell staining is a .ery .ersatile techni8ue an#0 if the antigen is highly
locali-e#0 can #etect as fe, as a thousan# antigen molecules in a cell. 'n some circumstances0
cell staining may also *e use# to #etermine the appro1imate concentration of an antigen0
especially *y an image analy-er.
(ethods
$here are many metho#s to o*tain immunological #etection on tissues0 inclu#ing those tie#
#irectly to primary anti*o#ies or antisera. ( #irect metho# in.ol.es the use of a #etecta*le tag
e.g.0 fluorescent molecule0 gol# particles0 etc.0 " #irectly to the anti*o#y that is then allo,e# to
*in# to the antigen e.g.0 protein" in a cell.
(lternati.ely0 there are many in!i$ect met1o!". 'n one such metho#0 the antigen is *oun# *y a
primary anti*o#y ,hich is then amplifie# *y use of a secon#ary anti*o#y ,hich *in#s to the
primary anti*o#y. Ne1t0 a tertiary reagent containing an en-ymatic moiety is applie# an# *in#s
to the secon#ary anti*o#y. Khen the 8uaternary reagent0 or su*strate0 is applie#0 the en-ymatic
en# of the tertiary reagent con.erts the su*strate into a pigment reaction pro#uct0 ,hich
pro#uces a
color many colors are possi*le5 *ro,n0 *lack0 re#0 etc.0" in the same location that the original
primary anti*o#y recogni-e# that antigen of interest.
Some e1amples of "u%"t$ate" use# also kno,n as chromogens" are (EC %3(mino3>3
EthylCar*a-ole"0 or D(B %0%F3Diamino*en-i#ine". 9se of one of these reagents after e1posure
to the necessary en-yme e.g.0 horsera#ish pero1i#ase conjugate# to an anti*o#y reagent"
pro#uces a positi.e immunoreaction pro#uct. 'mmunocytochemical .isuali-ation of specific
antigens of interest can *e use# ,hen a less specific stain like 2)E 2emato1ylin an# Eosin"
cannot *e use# for a #iagnosis to *e ma#e or to pro.i#e a##itional pre#icti.e information
regar#ing treatment in some cancers0 for e1ample".
(lternati.ely the secon#ary anti*o#y may *e co.alently linke# to a fluorophore F'$C an#
Rho#amine are the most common" ,hich is #etecte# in a fluorescence or confocal microscope.
$he location of fluorescence ,ill .ary accor#ing to the target molecule0 e1ternal for mem*rane
proteins0 an# internal for cytoplasmic proteins. 'n this ,ay immunofluorescence is a po,erful
techni8ue ,hen com*ine# ,ith confocal microscopy for stu#ying the location of proteins an#
#ynamic processes e1ocytosis0 en#ocytosis0 etc.".
Ce## A$$ay Immunocytoc1emi"t$y +$otoco#
-ote: Do not let the tissues #ry out once they are re3hy#rate.
9se separate tu*s for anti*o#ies an# negati.e control sli#es to a.oi# contamination.

MATE-IALS

Cell (rray Sli#e
Co.erslips
Sli#e racks
Staining #ishes ,ith li#s
:lastic sli#e tray
/r*ital shaker
$ransfer pipettes

Deioni-e# ,ater D' 2
I
/"
:BS :hosphate Buffere# Saline"
$riton X3<BB
2y#rogen pero1i#e 2
I
/
I
"
:rimary anti*o#y
Biotinylate# secon#ary anti*o#y0 2R: conjugate#
Bo.ine Serum (l*umin BS( A for *locking"
Strepta.i#in32R:
D(B
2emato1ylin optional"
(cetic (ci# optional"
;lycerol

+e$mea%i#i;e Mem%$ane /ptional if #etecting a mem*rane protein."

<. (## one #rop of :BS+B.<Z $riton X3<BB to each ,ell to permea*ili-e the cells. 'ncu*ate
sli#es for one <" minute at room temperature.
I. Remo.e the li8ui# an# ,ash the sli#es t,ice I1" in :BS0 L minutes each on the shaker.
%. Remo.e the li8ui# an# place the sli#es onto a tray.

B#oc5ing

@. Soak sli#es in <.LZ 2
I
/
I
+:BS solution for <L minutes.
L. Kash t,ice I1" in :BS for L minutes each on the shaker.
E. 'ncu*ate ,ith LZ BS( into each ,ell to *lock for o.ernight at @bC in a humi# cham*er.

+$ima$y Anti%o!y

?. Dilute the primary anti*o#y to the recommen#e# concentration in <Z BS( #iluent.
=. Remo.e BS( from the sli#es.
>. (## %La! of primary anti*o#y to each ,ell. 'ncu*ate for one <" hour at room
temperature.
<B. Remo.e the primary anti*o#y solution an# ,ash sli#es three %" times in :BS0 L minutes
each on the shaker.

Secon!a$y Anti%o!y an! 4etection

<<. Dilute the *iotinylate# secon#ary anti*o#y to <:IBB in a solution of <Z BS( #iluent.
<I. Remo.e the e1cess flui# an# a## one #rop secon#ary anti*o#y solution into each ,ell.
'ncu*ate for one <" hour at room temperature.
<%. Kash in :BS three %" times L minutes each on an or*ital shaker. Remo.e e1cess flui#.
<@. (## one #rop strepta.i#in32R: to each ,ell. 'ncu*ate for %B minutes at room
temperature.
<L. Kash three %" times L minutes in :BS on an or*ital shaker. Remo.e e1cess flui#.
<E. (## D(B solution to each cell ,ell. /nce the cells start turning *ro,n ine1perience#
technicians may ,ish to o*ser.e this un#er a microscope" ,ash t,ice I1" in :BS for L
minutes each time on the shaker.

Otiona# Counte$"tain

<?. Dip the sli#e rack ,ith the sli#es into a staining #ish of hemato1ylin for %B secon#s.
<=. Remo.e an# place into an aci# *ath IBBm! D' 2
I
/ an# one to three #rops of acetic
aci#". Rinse ,ith D' 2
I
/.

Co'e$ S#i"

<>. (## se.eral #rops of co.erslip solution LBZ glycerol + D' 2
I
/" to the sli#e.
IB. :lace the co.erslip on top of the sli#e.
I<. Store sli#es at room temperature.
Immuno*#uo$e"cence
Microphotograph of a histological section of human skin prepare# for !i$ect
immuno*#uo$e"cence using an anti3'g( anti*o#y. $he skin is from a patient ,ith 2enoch3
Schonlein purpura: 'g( #eposits are foun# in the ,alls of small superficial capillaries yello,
arro,s". $he pale ,a.y green area on top is the epi#ermis0 the *ottom fi*rous area is the #ermis.
Microphotograph of a histological section of human skin prepare# for !i$ect
immuno*#uo$e"cence using an anti3'g; anti*o#y. $he skin is from a patient ,ith systemic lupus
erythematosus an# sho,s 'g; #eposit at t,o #ifferent places: $he first is a *an#3like #eposit
along the epi#ermal *asement mem*rane 4lupus *an# test4 is positi.e". $he secon# is ,ithin the
nuclei of the epi#ermal cells anti3nuclear anti*o#ies".
Immuno*#uo$e"cence is a techni8ue use# for light microscopy ,ith a fluorescence microscope
an# is use# primarily on *iological samples. $his techni8ue uses the specificity of anti*o#ies to
their antigen to target fluorescent #yes to specific *iomolecule targets ,ithin a cell0 an#
therefore allo,s .isualisation of the #istri*ution of the target molecule through the sample.
'mmunofluorescence is a ,i#ely use# e1ample of immunostaining an# is a specific e1ample of
immunohistochemistry that makes use of fluorophores to .isualise the location of the anti*o#ies.
6<7
'mmunofluorescence can *e use# on tissue sections0 culture# cell lines0 or in#i.i#ual cells0 an#
may *e use# to analyse the #istri*ution of proteins0 glycans0 an# small *iological an# non3
*iological molecules. 'mmunofluoresence can *e use# in com*ination ,ith other0 non3anti*o#y
metho#s of fluorescent staining0 for e1ample0 use of D(:' to la*el DN(. Se.eral microscope
#esigns can *e use# for analysis of immunofluorescence samples5 the simplest is the
epifluorescence microscope0 an# the confocal microscope is also ,i#ely use#. &arious super3
resolution microscope #esigns that are capa*le of much higher resolution can also *e use#.
6I7
Types of immunofluorescence
$here are t,o classes of immunofluorescence techni8ues0 primary or #irect" an# secon#ary or
in#irect".
+$ima$y (!i$ect)
:rimary0 or #irect0 immunofluorescence uses a single anti*o#y that is chemically linke# to a
fluorophore. $he anti*o#y recognises the target molecule an# *in#s to it0 an# the fluorophore it
carries can *e #etecte# .ia microscope. $his techni8ue has se.eral a#.antages o.er the
secon#ary or in#irect" protocol *elo, *ecause of the #irect conjugation of the anti*o#y to the
fluorophore. $his re#uces the num*er of steps in the staining proce#ure0 is therefore faster0 an#
can a.oi# some issues ,ith anti*o#y cross3reacti.ity or non3specificity0 ,hich can lea# to
increase# *ackgroun# signal.
Secon!a$y (in!i$ect)
Secon#ary0 or in#irect0 immunofluorescence uses t,o anti*o#ies5 the first the primary anti*o#y"
recognises the target molecule an# *in#s to it0 an# the secon# the secon#ary anti*o#y"0 ,hich
carries the fluorophore0 recognises the primary anti*o#y an# *in#s to it. $his protocol is more
comple1 than the primary or #irect" protocol a*o.e an# takes more time *ut allo,s more
fle1i*ility.
$his protocol is possi*le *ecause an anti*o#y consists of t,o parts0 a .aria*le region ,hich
recogni-es the antigen" an# an in.ariant region ,hich makes up the structure of the anti*o#y
molecule". ( researcher can generate se.eral primary anti*o#ies that recogni-e .arious antigens
ha.e #ifferent .aria*le regions"0 *ut all share the same in.ariant region. (ll these anti*o#ies
may therefore *e recogni-e# *y a single secon#ary anti*o#y. $his sa.es the cost of mo#ifying
the primary anti*o#ies to #irectly carry a fluorophore.
Different primary anti*o#ies ,ith #ifferent in.ariant regions are typically generate# *y raising
the anti*o#y in #ifferent species. For e1ample0 a researcher might create primary anti*o#ies in a
goat that recogni-e se.eral antigens0 an# then employ #ye3couple# ra**it secon#ary anti*o#ies
that recogni-e the goat anti*o#y in.ariant region 4ra**it anti3goat4 anti*o#ies". $he researcher
may then create a secon# set of primary anti*o#ies in a mouse that coul# *e recognise# *y a
separate 4#onkey anti3mouse4 secon#ary anti*o#y. $his allo,s re3use of the #ifficult3to3make
#ye3couple# anti*o#ies in multiple e1periments.
0imitations
(s ,ith most fluorescence techni8ues0 a significant pro*lem ,ith immunofluorescence is
photo*leaching. !oss of acti.ity cause# *y photo*leaching can *e controlle# *y re#ucing the
intensity or time3span of light e1posure0 *y increasing the concentration of fluorophores0 or *y
employing more ro*ust fluorophores that are less prone to *leaching e.g.0 (le1a Fluors0 Seta
Fluors0 or Dy!ight Fluors".
'n general0 immunofluorescence is limite# to fi1e# i.e.0 #ea#" samples. (nalysis of structures
,ithin li.e cells *y immunofluorescence is not possi*le0 as anti*o#ies cannot cross the cell
mem*rane. (s such some uses of immunofluorescence ha.e *een outmo#e# *y the #e.elopment
of recom*inant proteins containing fluorescent protein #omains0 e.g.0 green fluorescent protein
;F:". 9se of such 4tagge#4 proteins allo,s #etermination of their localisation in li.e cells.
5mmunofluorescence (ethod
$he purpose of immunofluorescence is to #etect the location an# relati.e a*un#ance of any
protein for ,hich you ha.e an anti*o#y. /nce you ha.e anti*o#ies to your fa.orite protein0 you
can use them to in#icate ,here the protein is locate#. 'n this e1ample0 ,e ,ill use anti*o#ies for
the ca#cium AT+a"e0 or pump0 that is locate# in the en!o#a"mic $eticu#um ER" of .ery cell.
$he anti*o#y use# here only recogni-e# the chicken calcium ($:ase *ut immunofluorescence
can *e use# on any protein.
$he key to this entire process is the a*ility to .isuali-e the anti*o#y ,hen looking through a
microscope. Since anti*o#ies are smaller than calcium ($:ases0 you cannot see the anti*o#y
#irectly. $herefore0 you ha.e to use a fluorescent #ye that is co.alently attache# to the anti*o#y.
Khen a light illuminates the fluorescent #ye0 it a*sor*s the light an emits a #ifferent color light
,hich is .isi*le to the in.estigator an# can *e photographe#.
,igu$e ?3 'n most immunofluorescence e1periments0 t,o anti*o#ies are employe#. $he first one0
calle# the $ima$y anti%o!y0 is typically generate# in a mouse an# *in#s to your fa.orite
protein0 ,hich in this case is the chicken calcium ($:ase sho,n as a series un#ulating stripe#
line that -ig-ags through teh ER mem*rane <B times". $he "econ!a$y anti%o!y ,as purchase#
from a company that sells anti*o#ies that *in# to mouse anti*o#ies an# ha.e a fluorescent #ye
co.alently attache# to it. (s illustrate# here0 the secon#ary anti*o#ies can *in# to multiple sites
on the primary anti*o#y an# thus pro#uce a *righter signal since more #yes are *rought to a
single location. $he first step is to choose your cells of interest. 'n this case0 ,e ,ill look at a
chicken fi*ro*last0 or skin cell. 't ,as gro,n in tissue culture an# so it appears as an isolate# cell
,itn no .isi*le neigh*ors. $he cell ,as fi1e# ,ith formal#ehy#e to retain the shape an# location
of all cellular proteins. $he cell ,as treate# ,ith a mil# #etergent to #isol.e small holes in the
mem*ranes so the anti*o#ies coul# ha.e access to the cytoplasm. Because the calcium ($:ase is
locate# in the ER0 the anti*o#ies must ha.e access to the cytoplasm or they coul# not *in# to the
target protein.
,igu$e 23 $his immunofluorescence micrograph sho,s the ER *eing la*ele# ,ith a monoclonal
anti*o#y against the the chicken calcium ($:ase. $his chicken cell ,as fi1e#0 permea*lili-e#0
an# processe# for immunofluorescence. Khite in#icates the location of the fluorescent anti*o#y
an# thus the calcium ($:ase to ,hich the anti*o#y ,as *oun#. 'mmunofluorescence
photomicrograph *y (. Malcolm Camp*ell.
Immuno En;yme Tec1ni.ue" in Cytoc1emi"t$y
Summa$y
$he intro#uction of light an# especially electron optic systems for morphological stu#ies of
cellular an# su*cellular structures has ena*le# significant a#.ances to *e ma#e in the kno,3
le#ge of cell *iology an# normal an# #isease# organs.2o,e.er0 spatial an# temporal aspects of
cellular processes an# the functional or e.olutionary significance of the increasing comple31ity
of higher organisms cannot *e eluci#ate# simply *y comparing fine structures. 'n this conte1t0
classical histochemistry an# ne, specific cytological proce#ures un#er #e.elopment allo,
relationships *et,een *iological structure an# function to *e more rea#ily #iscerne#. 'n or#er to
un#erstan# the molecular composition of organs at the cellular le.el0 the com*ina3tion of
immunological an# histological concepts is a promising line of research ,hich alrea#y pro.e#
e1tremely useful for histopathology an# cell *iology. (nti*o#ies possess a high #egree of
specificity to,ar#s antigenic #eterminants. Because of the narro, range of specificity of an
anti*o#y molecule to *in# ,ith its antigenic #eterminant0 immunochemical metho#s are part of
the most sensiti.e techni8ues in molecular *iology an# *iome#icine. Kith respect to the
#efinition of antigenic molecules su*stances ,hich initiate the formation of an# react ,ith
anti*o#ies are calle# antigens"0 immunoserological analyses of organs of normal state an# in
#isease are of great importance. $o this aim0 8ualitati.e an# 8uantitati.e approaches ha.e *een
#escri*e# since the .ery early years of this century e.g. *y Ehrlich0 !an#steiner0 Kite*s3ky0
2ei#el*erger0 Marrack0 Na*at0 /u#in0 ;ra*ar an# schools #eri.e# from these pioneers in
immunochemistry.
'n our #ay0 further #e.elopments of highly sensiti.e techni8ues like those *ase# on ra#io3 or
en-yme3immuno3assays are still in progress. $he principle of an immunoserological analysis of
organs relies on the use of immune sera pro#uce# *y heteroimmuni-ation of animals0 on the use
of anti*o#ies pro#uce# *y hy*ri#omas or other *iotechnical proce#ures or on the occurrence of
autoanti*o#ies in connection ,ith certain #iseases. ( num*er of phenomena ,hich resem*le
anti*o#y reaction are share# *y lectins. $hese occur in a .ariety of plants0 in.erte*rates an#
.erte*rates an# are use# for the stu#y of car*ohy#rate moieties.
$he immunofluorescent approach intro#uce# *y Coons an# co3,orkers <>@<0 <>LB" opene#
specific in.estigations on cellular structure an# function at the light microscopic le.el. 'n the
meantime0 immunofluorescent metho#s ha.e progresse# from pure scientific research to,ar#s
routine in histopathology. 't is e.i#ent that analogous techni8ues ,ill also *e useful an#
important for ultrastructural stu#ies.
'n principle0 the resolution of the electron microscope ena*les the #emonstration of an anti3*o#y
molecule ,hich has reacte# ,ith its antigen. 2o,e.er0 after usual resin em*e#ment single
protein molecules in the tissue cannot *e i#entifie# *ecause such molecule groups are not more
electron #ense than the surroun#ing matri1. 'n conse8uence0 unla*ele# anti*o#ies are only
suita*le for the #emonstration of isolate# particles ,hen measura*le an# repro#uci*le changes in
#ensity or #efinite structural changes are o*taine#.
$he purpose of most immunohistological proce#ures is the i#entification an# characteri-ation of
cellular structure or function in situ rather than immuno3staining of physicochemically iso3late#
constituents. 2ence0 the respecti.e immunological ligan# must *e Ola*ele#P in a ,ay so that the
antigen3anti*o#y comple1es *ecome rea#ily .isi*le. Suita*le su*stances for la*eling purposes
are those ,hich lea# to #istinct color+fluorescent reactions e.g. light or fluorescent microscopes0
laser scanning microscopes" or ,hich gi.e significant #eflection of electrons in the electron
microscope. ( milestone in immuno3electron microscopy ,as then the conjuga3tion of the
metalloprotein ferritin ,ith anti*o#ies *y Singer <>L>" ,hich opene# a ne, era of
ultrastructure research.
't is no, ,ell esta*lishe# that immunological concepts of cellular ligan# assays at *oth light an#
electron microscopic le.els are important for the stu#y of histogenesis0 histo#ifferentia3tion an#
histopathology of organs. 'n this pu*lication0 the major steps in preparation of immu3
nohistological reagents on the one han# an# tissue sampling on the other han# are #escri*e#.
$he #etection of intracellular molecules is especially emphasi-e# ,hich is much more intri3cate
than that of e1tracellular spaces an# cell surface mem*ranes. $he use of soli# tissues instea# of
single cell suspensions or monolayer cultures is preferentially treate# for the reason that tissues
or their fragments represent the majority of specimens in histopathology an# that0 accor#ing to
current e1perience0 pitfalls are mainly o*ser.e# ,ith such soli# organ prepara3tions. 'n any case0
principles in the preparation of immunohistological reagents are the same an# theoretical as ,ell
as practical consi#erations of tissue sampling are 8uite similar for *oth tissue fragments an#
single cells.
Khen cells are to *e stu#ie#0 preser.ation of their structure an# minimal alteration from the
li.ing state shoul# *e consi#ere#. $he a#aption of a fi1ation metho# is in most cases necessa3ry.
Jet0 one of the most limiting factors impe#ing full utili-ation of immunological reagents is
fi1ation an# em*e#ment of *iological specimens e.g. em*e#ment in epo1y resins". Numerous
pu*lications #uring the last #eca#es ha.e sho,n that *y e1perimental testing an# metho#olo3
gical impro.ement of immunochemical an# cytological parameters0 con#itions can *e o*tai3ne#
un#er ,hich suita*le intracellular la*eling of cellular molecules is o*taine#.
( general an# i#eal proce#ure for the #etection of cellular ligan#s is not a.aila*le. !ight an#
electron microscopic immuno3stainings must *e usually esta*lishe# for each *iological mo#el.
$hus0 immunohistology may consi#er t,o particular parts:
o the preparation of immunohistological reagents as ,ell as
o the cytological assays.
'n this ,ay0 at least t,o #ifferent areas meet together0 namely immunochemistry e.en if
numerous reagents can *e no, purchase#" an# cellular morphology.
,ig3 ?2 Ste" o* ti""ue $oce""ing in $eem%e!ment immuno1i"to#ogy *$om incu%ation to
Eon em%e!ment3 Fro-en cut sections are immuno3staine#0 ,ashe#0 #ehy#rate# an# infiltrate#
,ith em*e##ing mi1ture in small glass .ials right". $hen0 single sections are place# into
polyethylene li#s from Beem capsules or e8ui.alent containing a #roplet of fresh Epon mi1ture
mi##le". From polymeri-e# Epon mol#s ,hich are prepare# in a#.ance in li#s of Beem
capsules0 Epon is remo.e# an# put ,ith the flat surface on top of the section. (fter su*se8uent
polymeri-ation a #esire# part of the section is selecte# *y light microscopy. $hen0 tissue an#
Epon e1cess are cut a,ay ,ith a ra-or *la#e. $he preparation is mounte# ,ith a#hesi.e or
Epon" on a casule fille# ,ith Epon an# poly3meri-e# in a#.ance". Finally0 the selecte# tissue
area of the flat em*e##e# section is trimme# for ultramicrotomy left".

,ig3 2a6!2 Com#ete 40 Lm t1ic5 *$o;en "ection a*te$ immuno6"tainingE !e1y!$ation an!
Eon em%e!ment ("ee ,ig3 ?)A a6c) "e'e$a# #ig1t mic$o"coic 'ie:" o* an a$ea o* inte$e"t
(ma$5e! :it1 a $a;o$ %#a!e) :1ic1 i" "e#ecte! *o$ *u$t1e$ "tu!ie"A !) "emit1in "ection o*
"ame $ea$ation3 +o"iti'e immuno6"taining" in c) an! !) a$e in!icate! %y a$$o:"3
,ig3 @a6c2 T$an"'e$"e "ec6tion" o* 40Lm t1ic5 *$o;en "ection"E $ea$e! a" !e"6c$i%e! in ,ig3
? an! ,ig3 23 Note o"iti'e immuno6"taining an! goo! ene6t$ation o* immunocytoc1e6
mica# $eagent" ac$o"" t1e 40 Lm t1ic5 *$o;en "ection"3 ,ig3 @c $e$e"ent" an immu6
nocyto#ogica# cont$o# incu6%ation3
,ig3 4a6%2 4etection o* in6t$ace##u#a$ IgG %y u"e o* anti%o!y6H-+ con0ugate" in
$eem%e!ment immuno61i"to#ogyA a) u#t$at1in "ec6tionE note immuno6"taining o* "ome
-E- #ame##aeA %) one Lm t1ic5 Eon "ection o* t1e "ame ti""ue in t1e e#ect$on mic$o"coeE
note t1e inte$connecte! "y"tem o* "taine! -E- #ame##ae3
Immuno ,e$$tin Tec1ni.ue"
Immuno*e$$itin La%e##ing
$his techni8ue is an a#aptation of the la*elling principle of fluorescent anti*o#y staining. 'n this
metho# of the anti*o#y is conjugate# to an electron#ense molecule0 such as ferritin0 an# is ma#e
.isi*le0 un#er the electron microscope. Ferritin is an organic comple1 of ferric hy#ro1i#e an#
ferric phosphate associate# ,ith apoferritin0 a protein of the li.er an# spleen that ser.es as a
storage place of iron. Cells are incu*ate# ,ith ferritin3conjugate# anti*o#y. Cell sections are
then e1amine# un#er the election microscope. Ferritin molecules can *e o*ser.e# on the cell
surface ,here anti*o#ies are *oun#.
( general metho# for the ultrastructural locali-ation of intracellular proteins an# antigens *y
immunoferritin techni8ues has *een #e.elope#. $he metho# in.ol.es #irect staining of ultrathin
sections of mil#ly glutaral#ehy#e3 fi1e# an# fro-en tissues cut *y means of a cryo3
ultramicrotome. Bo.ine pancreatic sections ,ere cut0 mounte# on gri#s0 an# staine# ,ith
ferritin3ra**it anti*o.ine RNase conjugates. (fter negati.e staining ,ith B.IZ
phosphotungstic aci#0 electron micrographs re.eale# specific la*eling of all of the -ymogen
granules an# the cisternae of the rough en#oplasmic reticulum. No significant
la*eling ,as seen in the nucleus0 mitochon#ria0 or cell sap regions. $he o*ser.ation that no
significant la*eling ,as foun# in any region of rat pancreatic sections ,as consistent
,ith the fact that rat RNase is immunologically noncrossreacti.e ,ith *o.ine RNase. 'n
a##ition0 the la*eling seen in *o.ine pancreas ,as completely a*sent if the sections
,ere first incu*ate# ,ith free anti*o#y. $he metho# use# here a.oi#s prolonge# fi1ation0
#ehy#ration0 an# other harsh chemical or physical treatments0 an# shoul# e1ten# the usefulness
of immunoferritin techni8ues to the intracellular locali-ation of many protein antigens *eyon#
pre.iously a.aila*le metho#s.
Immuno E#ect$on Mic$o"coy
$his stu#y e.aluate# a .ariety of fi1ati.es an# metho#s of
$his stu#y e.aluate# a .ariety of fi1ati.es an# metho#s of tissue preparation for application cf
the #irect pero1i#ase3la*ele# anti*o#y techni8ue to rat ki#ney specimens. $issue ultrastructure
,as most satisfactorily preser.e# an# the antigens stu#ie# ra**it 'g;0 human 'g;0 an# rat
$amm32orsfall protein $2:"" ,ere a#e8uately preser.e# after *rief fi1ation ,ith <Z
glutaral#ehy#e <L mm"0 a mi1ture of paraformal#ehy#e l?c" an# glutaral#ehy#e B.BLZ"0 or a
paraformal#ehy#e0 lysine0 perio#ate fi1ati.e. ;lycerol su*stitution ,as consi#ere#
an important step ,hich minimi-e# ice crystal artifacts. Free-ing an# tha,ing ,ere essential
steps that facilitate# a#e8uate penetration of la*ele# anti*o#y to p specific antigenic sites. $he
#istri*ution of injecte# ra**it 'g; anti3rat glomerular *asement mem*rane ;BM" anti*o#y"
,as pre#ominately on the lamina rara interna an# e1terna of the ;BM. 'njecte# aggregate#
human 'g; p ,as foun# primarily ,ithin the spaces *et,een glomeruplar mesangial cells. Ra**it
anti3rat $2: ,as locali-e# primarily on the infol#ing mem*ranes of cells of the ascen#ing thick
lim* of 2enle. Ke suggest that the metho#s #escri*e# may ha.e ,i#e application.
UNIT 7 MOLECULA- IMMUNOLOGY
7accine
( 'accine is a *iological preparation that impro.es immunity to a particular #isease. ( .accine
typically contains an agent that resem*les a #isease3causing microorganism0 an# is often ma#e
from ,eakene# or kille# forms of the micro*e or its to1ins. $he agent stimulates the *o#yFs
immune system to recogni-e the agent as foreign0 #estroy it0 an# 4remem*er4 it0 so that the
immune system can more easily recogni-e an# #estroy any of these microorganisms that it later
encounters.
&accines can *e prophylactic e.g. to pre.ent or ameliorate the effects of a future infection *y
any natural or 4,il#4 pathogen"0 or therapeutic e.g. .accines against cancer are also *eing
in.estigate#5 see cancer .accine".$he term !accine #eri.es from E#,ar# VennerFs <?>E use of
the term co po' !atin !ariol. !accin.0 a#apte# from the !atin !acc/n-us0 from !acca co,"0
,hich0 ,hen a#ministere# to humans0 pro.i#e# them protection against smallpo1.
History
Sometime #uring the <??Bs E#,ar# Venner hear# a milkmai# *oast that she ,oul# ne.er ha.e
the often3fatal or #isfiguring #isease smallpo10 *ecause she ha# alrea#y ha# co,po10 ,hich has
a .ery mil# effect in humans. 'n <?>E0 Venner took pus from the han# of a milkmai# ,ith
co,po10 inoculate# an =3year3ol# *oy ,ith it0 an# si1 ,eeks later .ariolate# the *oyFs arm ,ith
smallpo10 after,ar#s o*ser.ing that the *oy #i# not catch smallpo1. Further e1perimentation
#emonstrate# the efficacy of the proce#ure on an infant. Since .accination ,ith co,po1 ,as
much safer than smallpo1 inoculation0 the latter0 though still ,i#ely practice# in Englan#0 ,as
*anne# in <=@B. !ouis :asteur generali-e# VennerFs i#ea *y #e.eloping ,hat he calle# a ra*ies
.accine0 an# in the nineteenth century .accines ,ere consi#ere# a matter of national prestige0
an# compulsory .accination la,s ,ere passe#.
$he t,entieth century sa, the intro#uction of se.eral successful .accines0 inclu#ing those
against #iphtheria0 measles0 mumps0 an# ru*ella. Major achie.ements inclu#e# the #e.elopment
of the polio .accine in the <>LBs an# the era#ication of smallpo1 #uring the <>EBs an# <>?Bs.
Maurice 2illeman ,as the most prolific of the #e.elopers of the .accines in the t,entieth
century. (s .accines *ecame more common0 many people *egan taking them for grante#.
2o,e.er0 .accines remain elusi.e for many important #iseases0 inclu#ing malaria an# 2'&.
Types
(.ian flu .accine #e.elopment *y re.erse genetics techni8ues.
&accines are #ea# or inacti.ate# organisms or purifie# pro#ucts #eri.e# from them.
$here are se.eral types of .accines currently in use. $hese represent #ifferent strategies use# to
try to re#uce risk of illness0 ,hile retaining the a*ility to in#uce a *eneficial immune response.
?3 Hi##e!
Some .accines contain kille#0 *ut pre.iously .irulent0 micro3organisms that ha.e *een #estroye#
,ith chemicals or heat. E1amples are the influen-a .accine0 cholera .accine0 *u*onic plague
.accine0 polio .accine0 hepatitis ( .accine0 an# ra*ies .accine.
23 Attenuate!
Some .accines contain li.e0 attenuate# microorganisms. Many of these are li.e .iruses that ha.e
*een culti.ate# un#er con#itions that #isa*le their .irulent properties0 or ,hich use closely3
relate# *ut less #angerous organisms to pro#uce a *roa# immune response5 ho,e.er0 some are
*acterial in nature. $hey typically pro.oke more #ura*le immunological responses an# are the
preferre# type for healthy a#ults. E1amples inclu#e the .iral #iseases yello, fe.er0 measles0
ru*ella0 an# mumps an# the *acterial #isease typhoi#. $he li.e Myco*acterium tu*erculosis
.accine #e.elope# *y Calmette an# ;uWrin is not ma#e of a contagious strain0 *ut contains a
.irulently mo#ifie# strain calle# 4BC;4 use# to elicit immunogenicity to the .accine.
@3 To&oi!
$o1oi# .accines are ma#e from inacti.ate# to1ic compoun#s that cause illness rather than the
micro3organism. E1amples of to1oi#3*ase# .accines inclu#e tetanus an# #iphtheria. $o1oi#
.accines are kno,n for their efficacy. Not all to1oi#s are for micro3organisms5 for e1ample0
0rotalus atro' to1oi# is use# to .accinate #ogs against rattlesnake *ites.
Su%unit
:rotein su*unit A rather than intro#ucing an inacti.ate# or attenuate# micro3organism to an
immune system ,hich ,oul# constitute a 4,hole3agent4 .accine"0 a fragment of it can create an
immune response. E1amples inclu#e the su*unit .accine against 2epatitis B .irus that is
compose# of only the surface proteins of the .irus pre.iously e1tracte# from the *loo# serum of
chronically infecte# patients0 *ut no, pro#uce# *y recom*ination of the .iral genes into yeast"0
the .irus3like particle &!:" .accine against human papilloma.irus 2:&" that is compose# of
the .iral major capsi# protein0 an# the hemagglutinin an# neuramini#ase su*units of the
influen-a .irus.
Con0ugate
Conjugate A certain *acteria ha.e polysacchari#e outer coats that are poorly immunogenic. By
linking these outer coats to proteins e.g. to1ins"0 the immune system can *e le# to recogni-e the
polysacchari#e as if it ,ere a protein antigen. $his approach is use# in the &aemophilus
influen*ae type B .accine.
E&e$imenta#
( num*er of inno.ati.e .accines are also in #e.elopment an# in use:
Den#ritic cell .accines com*ine #en#ritic cells ,ith antigens in or#er to present the
antigens to the *o#yFs ,hite *loo# cells0 thus stimulating an immune reaction. $hese
.accines ha.e sho,n some positi.e preliminary results for treating *rain tumors.
Recom*inant &ector A *y com*ining the physiology of one micro3organism an# the
DN( of the other0 immunity can *e create# against #iseases that ha.e comple1 infection
processes
DN( .accination A in recent years a ne, type of .accine calle# $-+ !accination0
create# from an infectious agentFs DN(0 has *een #e.elope#. 't ,orks *y insertion an#
e1pression0 triggering immune system recognition" of .iral or *acterial DN( into human
or animal cells. Some cells of the immune system that recogni-e the proteins e1presse#
,ill mount an attack against these proteins an# cells e1pressing them. Because these
cells li.e for a .ery long time0 if the pathogen that normally e1presses these proteins is
encountere# at a later time0 they ,ill *e attacke# instantly *y the immune system. /ne
a#.antage of DN( .accines is that they are .ery easy to pro#uce an# store. (s of IBBE0
DN( .accination is still e1perimental.
$3cell receptor pepti#e .accines are un#er #e.elopment for se.eral #iseases using mo#els
of &alley Fe.er0 stomatitis0 an# atopic #ermatitis. $hese pepti#es ha.e *een sho,n to
mo#ulate cytokine pro#uction an# impro.e cell me#iate# immunity.
$argeting of i#entifie# *acterial proteins that are in.ol.e# in complement inhi*ition
,oul# neutrali-e the key *acterial .irulence mechanism.
Khile most .accines are create# using inacti.ate# or attenuate# compoun#s from micro3
organisms0 synthetic .accines are compose# mainly or ,holly of synthetic pepti#es0
car*ohy#rates or antigens.
7a#ence
&accines may *e mono!alent also calle# uni!alent" or multi!alent also calle# poly!alent". (
mono.alent .accine is #esigne# to immuni-e against a single antigen or single microorganism. (
multi.alent or poly.alent .accine is #esigne# to immuni-e against t,o or more strains of the
same microorganism0 or against t,o or more microorganisms. 'n certain cases a mono.alent
.accine may *e prefera*le for rapi#ly #e.eloping a strong immune response.
Developing immunity
$he immune system recogni-es .accine agents as foreign0 #estroys them0 an# 4remem*ers4
them. Khen the .irulent .ersion of an agent comes along the *o#y recogni-es the protein coat
on the .irus0 an# thus is prepare# to respon#0 *y <" neutrali-ing the target agent *efore it can
enter cells0 an# I" *y recogni-ing an# #estroying infecte# cells *efore that agent can multiply to
.ast num*ers.
Khen t,o or more .accines are mi1e# together in the same formulation0 the t,o .accines can
interfere. $his most fre8uently occurs ,ith li.e attenuate# .accines0 ,here one of the .accine
components is more ro*ust than the others an# suppresses the gro,th an# immune response to
the other components. $his phenomenon ,as first note# in the tri.alent Sa*in polio .accine0
,here the amount of serotype I .irus in the .accine ha# to *e re#uce# to stop it from interfering
,ith the 4take4 of the serotype < an# I .iruses in the .accine. $his phenomenon has also *een
foun# to *e a pro*lem ,ith the #engue .accines currently *eing researche#0 ,here the DEN3%
serotype ,as foun# to pre#ominate an# suppress the response to DEN3<0 3I an# 3@ serotypes.
&accines ha.e contri*ute# to the era#ication of smallpo10 one of the most contagious an# #ea#ly
#iseases kno,n to man. /ther #iseases such as ru*ella0 polio0 measles0 mumps0 chickenpo10 an#
typhoi# are no,here near as common as they ,ere a hun#re# years ago. (s long as the .ast
majority of people are .accinate#0 it is much more #ifficult for an out*reak of #isease to occur0
let alone sprea#. $his effect is calle# her# immunity. :olio0 ,hich is transmitte# only *et,een
humans0 is targete# *y an e1tensi.e era#ication campaign that has seen en#emic polio restricte#
to only parts of four countries (fghanistan0 'n#ia0 Nigeria an# :akistan".$he #ifficulty of
reaching all chil#ren as ,ell as cultural misun#erstan#ings0 ho,e.er0 ha.e cause# the anticipate#
era#ication #ate to *e misse# se.eral times.
Effectiveness
&accines #o not guarantee complete protection from a #isease.Sometimes0 this is *ecause the
hostFs immune system simply #oes not respon# a#e8uately or at all. $his may *e #ue to a
lo,ere# immunity in general #ia*etes0 steroi# use0 2'& infection0 age" or *ecause the hostFs
immune system #oes not ha.e a B cell capa*le of generating anti*o#ies to that antigen.
E.en if the host #e.elops anti*o#ies0 the human immune system is not perfect an# in any case
the immune system might still not *e a*le to #efeat the infection.
(#ju.ants are typically use# to *oost immune response. Most often aluminium a#ju.ants are
use#0 *ut a#ju.ants like s8ualene are also use# in some .accines an# more .accines ,ith
s8ualene an# phosphate a#ju.ants are *eing teste#. !arger #oses are use# in some cases for ol#er
people LBA?L years an# up"0 ,hose immune response to a gi.en .accine is not as strong.
$he efficacy or performance of the .accine is #epen#ent on a num*er of factors:
the #isease itself for some #iseases .accination performs *etter than for other #iseases"
the strain of .accine some .accinations are for #ifferent strains of the #isease"
,hether one kept to the timeta*le for the .accinations
some in#i.i#uals are 4non3respon#ers4 to certain .accines0 meaning that they #o not
generate anti*o#ies e.en after *eing .accinate# correctly
other factors such as ethnicity0 age0 or genetic pre#isposition
Khen a .accinate# in#i.i#ual #oes #e.elop the #isease .accinate# against0 the #isease is likely
to *e mil#er than ,ithout .accination.
6
$he follo,ing are important consi#erations in the effecti.eness of a .accination program:
6
<. careful mo#elling to anticipate the impact that an immuni-ation campaign ,ill ha.e on
the epi#emiology of the #isease in the me#ium to long term
I. ongoing sur.eillance for the rele.ant #isease follo,ing intro#uction of a ne, .accine
an#
%. maintaining high immuni-ation rates0 e.en ,hen a #isease has *ecome rare.
'n <>L= there ,ere ?E%0B>@ cases of measles an# LLI #eaths in the 9nite# States.Kith the help
of ne, .accines0 the num*er of cases #roppe# to fe,er than <LB per year me#ian of LE".'n early
IBB=0 there ,ere E@ suspecte# cases of measles. L@ out of E@ infections ,ere associate# ,ith
importation from another country0 although only <%Z ,ere actually ac8uire# outsi#e of the
9nite# States5 E% of these E@ in#i.i#uals either ha# ne.er *een .accinate# against measles0 or
,ere uncertain ,hether they ha# *een .accinate#.
Trends
&accine #e.elopment has se.eral tren#s
9ntil recently0 most .accines ,ere aime# at infants an# chil#ren0 *ut a#olescents an#
a#ults are increasingly *eing targete#.
Com*inations of .accines are *ecoming more common5 .accines containing fi.e or more
components are use# in many parts of the ,orl#.
Ne, metho#s of a#ministering .accines are *eing #e.elope#0 such as skin patches0
aerosols .ia inhalation #e.ices0 an# eating genetically engineere# plants.
&accines are *eing #esigne# to stimulate innate immune responses0 as ,ell as a#apti.e.
(ttempts are *eing ma#e to #e.elop .accines to help cure chronic infections0 as oppose#
to pre.enting #isease.
&accines are *eing #e.elope# to #efen# against *ioterrorist attacks such as anthra10
plague0 an# smallpo1.
(ppreciation for se1 an# pregnancy #ifferences in .accine responses 4might change the
strategies use# *y pu*lic health officials4.
:rinciples that go.ern the immune response can no, *e use# in tailor3ma#e .accines against
many noninfectious human #iseases0 such as cancers an# autoimmune #isor#ers.For e1ample0
the e1perimental .accine CJ$BBE3(ngR* has *een in.estigate# as a possi*le treatment for high
*loo# pressure.Factors that ha.e impact on the tren#s of .accine #e.elopment inclu#e progress
in translatory me#icine0 #emographics0 regulatory science0 political0 cultural0 an# social
responses.
Cu$$ent a$oac1e" to 'accine $ea$ation
Numerous con.entional .accines for animal use are currently a.aila*le0 an# many of these
.accines ha.e *een instrumental in the control of infectious #iseases of major economic
importance. ( .accine has e.en *een instrumental in glo*al era#ication of smallpo10 an
important human #isease. 2o,e.er0 many of the current .accines are #eficient in efficiency0
potency0 or safety. 't has *een recogni-e# that the con.entional metho#ologies are a limitation to
further .accine #e.elopment. 'ntro#uction of monoclonal anti*o#ies0 recom*inant DN(0 an#
protein engineering techni8ues has facilitate# a rather rapi# increase in the kno,le#ge of
pathogenetic mechanisms0 as ,ell as of protecti.e antigens at the molecular le.el. $his
kno,le#ge pro.i#es the *asis for #e.elopment of a ne, generation of .accines. (s a rule0 these
.accines contain purifie# immunogens0 or e.en isolate# epitopes0 i#entifie# an# prepare# *y
molecular *iological techni8ues. $he efforts to fin# *etter #eli.ery systems an# *etter a#ju.ants
accompany the research on .accines.
A#ication" o* -ecom%inant 4NA Tec1no#ogy
+o#yme$a"e C1ain -eaction (+C-) has a ,i#e range of applications in many
#isciplines
A Molecular Biology+Research
A Diagnostics
A ;enetic Counseling
A Criminology+Forensics
A :aternity testing
A (rcheology
A Foo# testing
A E.olutionary stu#ies
A!'antage" o'e$ t$a!itiona# met1o!o#ogie"
Fast an# efficient amplification of specific DN( se8uences
No re8uirement for cloning or su*cloning
$iny amounts of material are usually sufficient
Disease #iagnoses ,ill *e greatly e1pe#ite# *y :CR to i#entify microorganisms in
infecte# people ,ho ,oul# pro.e falsely negati.e *y other #iagnostic proce#ures
?3 4etection o* HI7 in T6#ym1ocyte"
Serological techni8ues reguire humoral immune responses to *ecome acti.ate#
for successful #etection of anti32'& anti*o#ies serocon.ersion"
(c8uire# immune responses can take <B3<@ #ays *efore (* titers reach ma1imum
le.els.
'n#i.i#ual may test negati.e an# transmit 2'& unkno,ingly False negati.e"
23 Human +ai##oma 7i$u" (H+7)
Causes genital ,arts an# cer.ical cancer
$issue sample from cer.i1 use# in pCR reaction
$reatment can *egin earlier
(cyclo.ir
;ancyclo.ir

A%;yme
(n a%;yme from anti*o#y an# en-yme"0 also calle# catmab from catalytic monoclonal
antibody"0 is a monoclonal anti*o#y ,ith catalytic acti.ity. Molecules ,hich are mo#ifie# to
gain ne, catalytic acti.ity are calle# syn-ymes. (*-ymes are usually artificial constructs0 *ut
are also foun# in normal humans anti3.asoacti.e intestinal pepti#e autoanti*o#ies" an# in
patients ,ith autoimmune #iseases such as systemic lupus erythematosus0 ,here they can *in#
to an# hy#roly-e DN(. (*-ymes are potential tools in *iotechnology0 e.g.0 to perform specific
actions on DN(.
En-ymes function *y lo,ering the acti.ation energy of the transition state0 there*y cataly-ing
the formation of an other,ise less3fa.ora*le molecular interme#iate *et,een reactants an#
pro#ucts. 'f an anti*o#y is #e.elope# to a sta*le molecule thatFs similar to an unsta*le
interme#iate of another potentially unrelate#" reaction0 the #e.elope# anti*o#y ,ill
en-ymatically *in# to an# sta*ili-e the interme#iate state0 thus cataly-ing the reaction. ( ne,
an# uni8ue type of en-yme is pro#uce#.
HI treatment
'n a Vune IBB= issue of the journal (utoimmunity Re.ie,s0 researchers S :lan8ue0 Su#hir :aul0
:h.D0 an# Jasuhiro Nishiyama0 :h.D of the 9ni.ersity /f $e1as Me#ical School at 2ouston
announce# that they ha.e engineere# an a*-yme that #egra#es the superantigenic region of the
gp<IB CD@ *in#ing site. $his is the one part of the 2'& .irus outer coating that #oes not change0
*ecause it is the attachment point to $ lymphocytes0 the key cell in cell3me#iate# immunity.
/nce infecte# *y 2'&0 patients pro#uce anti*o#ies to the more changea*le parts of the .iral coat.
$he anti*o#ies are ineffecti.e *ecause of the .irusF a*ility to change their coats rapi#ly. Because
this protein gp<IB is necessary for the 2'& .irus to attach0 it #oes not change across #ifferent
strains an# is a point of .ulnera*ility across the entire range of the 2'& .ariant population.
$he a*-yme #oes more than *in# to the site0 it actually #estroys the site0 ren#ering the 2'& .irus
inert0 an# then can attach to other .iruses. ( single a*-yme can #estroy thousan#s of 2'&
.iruses. 2uman clinical trials ,ill *e the ne1t step in pro#ucing treatment an# perhaps e.en
pre.entati.e .accines an# micro*ici#e.
A#ication O* +C- Tec1no#ogy To +$o!uce Anti%o!ie" An! Ot1e$ Immuno#ogica#
-eagent"
(nti*o#y pro#uction tests ha.e tra#itionally *een use# to test *iological materials for .iral
contamination.No, molecular *iology techni8ues ha.e emerge# as an alternati.e. $he authors
compare M(: testing ,ith :CR3*ase# #etection metho#s0 focusing on #ifferences in animal
use0 la*oratory re8uirements0 sample si-e0 an# limits of #etection.
Recently human anti*o#y genes can easily *e amplifie# *y :CR metho# from lymphocytes.
$hus0 genetically engineere# li*raries of human anti*o#ies ,ere prepare# *y using phage0 E.
coli an# animal cells as host cells. 't is reporte#0 ho,e.er0 that recom*inant %. cere!isiae coul#
secret human 'g; anti*o#y into fermentation *roth *ut faile# to secret human 'gM anti*o#y.
Ke firstly trie# to pro#uce a human 'gM3type anti*o#y *y secretion from recom*inant %.
cere!isiae. $he gene of a human anti3e1oto1in ( anti*o#y 'gM3type"0 ,hich coul# *e secrete#
from a human3mouse hy*ri#oma0 ,as use# as a mo#el anti*o#y gene. Ke also propose a ne,
metho# to o*tain a larger si-e of com*inatorial li*raries of anti*o#y *y use of t,o kin#s of
mating types in yeast cells0 a3type an# alpha3type. By transforming li*raries of genes of hea.y
an# light chains to a3 an# alpha3types respecti.ely0 for e1ample0 it shoul# *e possi*le to o*tain a
larger si-e of li*raries of anti*o#ies *y mating them. 'f ,e use hea.y an# light chain li*raries
containing <Bn kin#s of genes0respecti.ely0 a li*rary containing <BIn of anti*o#y shoul#
theoretically *e o*taine#. $he genes of hea.y an# light chains ,ere amplifie# *y :CR metho#
from the hy*ri#oma an# clone# on .ectors. Ke am#e a large si-e of a li*rary an# confirme# that
%. cere!isiae coul# secret the human3type anti*o#y into *roth.
Effecti.e pro#uction metho#s an# fermentation con#itions for recom*inant proteins *y secretion
form yeast cells ,ere also stu#ie#. Secretion of recom*inant proteins from P. pastoris can *e
in#uce# ,ith methanol *y use of (/X< promotor. $he effects of D/ an# the methanol
concentration of the pro#ucti.ity ,ere stu#ie#0 an# the secrete proteins ,ere characteri-e# *y
use of anti3pepti#e anti*o#ies.
Immuno T1e$ay 9it1 Genetica##y Enginee$e! Anti%o!ie"
E&$e""ion o* anti%o!y 1ea'y6 an! #ig1t6c1ain gene" %y
Genetica##y Enginee$e! Anti%o!ie"
E1pression of anti*o#y hea.y3 an# light3chain genes *y transfection permits the pro#uction of
monoctonal anti*o#ies ,ith impro.e# *iological an# antigen3*in#ing properties. $he
immunoglo*ulin genes are place# in .ectors containing a gene for enco#ing a protein that
pro.i#es a *iochemically selecta*le function in eukaryotic cells5 these .ectors are transfecte#
into myeloma an# hy*ri#oma cells. Selection of #rug3resistant cells permits the efficient
isolation of the rare cells that e1press the transfecte# DN(. By placing hea.y an#
light chains on plasmi#s ,ith #ifferent selecta*le markers0 one can #eli.er hea.y3 an# light3
chain genes simultaneously to the same cell. $he transfecte# immunoglo*ulin genes are
efficiently e1presse# an# the proteins pro#uce# are a faithful mirror of the genes that ,ere
intro#uce#. 9sing the stan#ar# techni8ues of genetic engineering an# gene transtection0 ,e
can no, pro#uce anti*o#ies of ,i#ely .arying structures0 inclu#ing chimeric anti*o#ies ,ith
segments #eri.e# from #ifferent species. $hese anti*o#ies pro.i#e useful reagents
to stu#y structure3function relationships ,ithin the anti*o#y molecule. 9ltimately it ,ill *e
possi*le to pro#uce a ne, generation of anti*o#y molecules ,ith impro.e# antigen*in#ing
properties an# effector functions.
E&am#e" *o$ Immuno T1e$ay 9it1 Genetica##y Enginee$e! Anti%o!ie"
AIM Recent progress in phage anti*o#y #isplay technology has re.olutioni-e# our a*ility to
select an# engineer human monoclonal anti*o#ies for therapy. (nti*o#y fragments ,ith
#esira*le specificities are selecte# from .ery large li*raries consisting of *illions of engineere#
phage particles each e1pressing an anti*o#y fragment ,ith a uni8ue specificity. /ur aim is to
e1ploit su*tracti.e phage selection metho#s to o*tain anti*o#y fragments that selecti.ely *in# to
tumor cells an# tumor3associate# en#othelial cells. 'n a##ition0 ,e ha.e e1ploite# this approach
to o*tain anti*o#y fragments that *in# to #isease3specific conformations of a non cell3*oun#
molecule. $hese anti*o#ies ha.e *een engineere# into fully human monoclonal anti*o#ies of
.arious isotypes an# affinities an# teste# in tumor mo#els for their therapeutic efficacy.
METHO4S2 Ke ha.e use# phage anti*o#y #isplay li*raries in com*ination ,ith flo,
cytometry to select for phages that *in# to intact leukemic or soli# tumor cells or to tumor3
associate# en#othelial cells from freshly o*taine# tumor samples. $hus0 anti*o#ies can *e
o*taine# against cells that ha.e not *een mo#ifie# *y in .itro cell culture. 'n a##ition0 the
proce#ure allo,s the isolation of anti*o#ies against .ery rare cells present in a heterogeneous
mi1ture. Furthermore0 ,e ha.e use# su*traction approaches to select for anti*o#ies that *in# to
the acti.e *ut not the nati.e conformation of .itronectin0 an e1tracellular matri1 protein in.ol.e#
in .arious patho"physiological processes0 inclu#ing cancer. (nti*o#ies ,ere teste# for their
specificity *y e1tensi.e flo, cytometric an# immunohistochemical analysis of normal an#
tumor tissues an# target antigens ,ere i#entifie# *y con.entional metho#s. (nti*o#y fragments
,ith rele.ant *in#ing patterns ,ere con.erte# into intact 'g;< or 'g( anti*o#ies an# analy-e#
for their functional acti.ities in .itro an# in .i.o assays. -ESULTS2 9sing phage #isplay
li*raries0 ,e ha.e generate# a panel of fully human monoclonal anti*o#ies that ha.e therapeutic
potential in the treatment of soli# an# leukemic tumors. $he anti*o#ies *in# to tumor3associate#
antigens e1presse# *y the tumor cells proper0 to antigens e1presse# *y tumor3associate#
en#othelial cells or to a tumor3associate# form of the e1tracellular matri1 protein .itronectin. (t
least in some cases0 ,e foun# that the anti*o#ies #i# not *in# to #e no.o e1presse# molecules
*ut rather to tumor3associate# conformations or glycosylation forms of molecules also e1presse#
on the mem*rane of non3tumor cells. Fully3human 'g( an# 'g; monoclonal anti*o#ies ha#
tumor cell killing properties *ase# on the recruitment of immunological effector cells an#
molecules. 'n a##ition0 some anti*o#ies #isplaye# a##itional tumor cell killing properties
in#epen#ent of immunological mechanisms. CONCLUSIONS2 :hage #isplay li*raries are a
source of human monoclonal anti*o#ies that *in# to tumor3associate# molecules an# that ha.e
potential therapeutic application. By using su*tracti.e phage selection metho#s0 anti*o#ies that
#etect su*tle alterations of molecules can *e o*taine# such as alterations in conformation or
glycosylation pattern. $hese approaches yiel# targets for tumor therapy that are not unco.ere#
*y genomics approaches
?3 A#ication o* genetica##y6enginee$e! anti6CEA anti%o!ie" *o$ otentia# immunot1e$ay
o* co#o$ecta# cance$3
2itherto anti3CE( monoclonal anti*o#ies M(*s"0 normally of mouse origin0 ha.e *een use#
primarily for clinical #iagnosis of colorectal cancer0 either as a tumor marker in serum to
monitor tumor recurrence0 or latterly as a means to locali-e in .i.o CE(3*earing tumors0 an#
metastases in patients. 'n .i.o #iagnosis using mouse anti3CE( M(*s has so far ha# limite#
clinical utility *ecause the anti*o#ies elicit a strong anti3mouse immunoglo*ulin immune
response on repeate# a#ministration in man. $his pro*lem has *een a##resse# *y the
#e.elopment of .arious strategies for 4humani-ation4 of mouse anti3CE( M(*s *y genetic
manipulation of immunoglo*ulin genes. Such humani-e#0 engineere# anti*o#ies marke#ly
attenuate the antigenic response #irecte# against the M(*0 such that safe0 repeate#
a#ministration to patients has *ecome feasi*le. Such humani-e# anti3CE( anti*o#ies can thus *e
ra#ioacti.ely3la*elle# an# applie# for in .i.o monitoring an# #etection of recurrent malignant
#isease0 or use# for therapeutic strategies ,hich similarly take a#.antage of the a*ility of the
anti*o#ies to target cytoto1ic agents selecti.ely to tumor cells. $he application of these no.el
proce#ures for manipulating M(* structure presents entirely ne, opportunities for #iagnosis
an# treatment of human colorectal cancer.
Human Gene T1e$ay
Genetica##y Enginee$e! Anti%o!ie" in Gene T$an"*e$ an! Gene T1e$ay
ABST-ACT
/ur a*ility to pro#uce an# engineer human monoclonal anti*o#ies pro.i#es a *asis for the
#e.elopment of no.el therapeutical strategies against a .ariety of #iseases. $hese strategies not
only inclu#e impro.e# passi.e immunotherapy *ut also more sophisticate# anti*o#y3*ase# gene
therapies in.ol.ing gene transfer approaches. Four of the major applications of anti*o#y gene
engineering in the fiel# of gene therapy are re.ie,e# here. $hese are <" the re#efinition of .iral
.ector tropism of infection for *etter trans#uction of cells of therapeutical interest0 I" the
grafting of ne, cell recognition acti.ities to effector cells of the immune system to kill cancer
an# pathogen3infecte# cells0 %" the inhi*ition of cellular an# .iral functions through intracellular
e1pression of anti*o#y3#eri.e# molecules0 an# @" the systemic #eli.ery of therapeutic
monoclonal anti*o#ies *y non3B cells in li.ing organisms.
O'e$'ie: "umma$y
Monoclonal anti*o#ies are potentially useful for the treatment of many #iseases. 'n this conte1t0
our a*ility to construct human monoclonal anti*o#ies has constitute# a major a#.ance to,ar#
the passi.e immuni-ation of human *eings. Moreo.er0 gene engineering not only permits the
cloning of immunoglo*ulin genes0 *ut also allo,s mo#ifications of .arious types. $hese inclu#e
impro.ement of kinetic+thermo#ynamic properties of anti*o#ies0 grafting of a##itional
*iological acti.ities0 an# #e.elopment of ne, classes of molecules. $his e1ten#s consi#era*ly
the scope of clinical applications of anti*o#ies. Research *y numerous groups ,orl#,i#e
in#icates that anti*o#y engineering may also pro.e a#.antageous in the fiel# of gene therapy.
(nti*o#ies an# anti*o#y3relate# molecules may *e use# either for #irecting gene transfer .ectors
o,ar# cells of therapeutic interest or as molecules ,ith intrinsinc therapeutic acti.ity.
Genetica##y enginee$e! anti%o!ie" *o$ !i$ect antineo#a"tic t$eatment an! "y"tematic
!e#i'e$y o* 'a$iou" t1e$aeutic agent" to cance$ ce##"
Classical antineoplastic therapeutic mo#alities such as surgery0 ra#iation0 an# chemotherapy not
only fail to cure the great majority of neoplasms0 *ut their employment often lea#s to se.ere an#
#e*ilitating si#e effects associate# ,ith se.ere neoplasm3relate# mor*i#ity. 'mmunotherapy as a
fourth mo#ality of anti3cancer therapy has alrea#y *een pro.en to *e 8uite effecti.e. $he
astonishing immunophenotypic ':" heterogeneity of neoplastic cells0 the #ifferent cytoto1ic
acti.ity associate# ,ith the moiety linke# to gi.en monoclonal anti*o#ies m(*"0 an# mostly the
impressi.e genetic mo#ulation capa*ilities of cancer cells still remain as yet unsol.e#
#ifficulties in the present immunotherapy of human neoplasms. $he a#.ances in m(*
pro#uction ha.e re.italise# the initial concept of use of cancer cell specific 4magic *ullets.4
(nti*o#ies represent ne, approaches to anti3cancer therapy: they are neoplastic cell3specific an#
lethal to neoplastically transforme# cells .ia immune effector mechanisms ,ith no to1icity to
normal tissues. $hey are *eing o*ser.e# an# #e.elope#0 a#hering to the ol# prayer: 4Destroy the
#isease# tissues0 preser.e the normal.4 Strategies for the employment of anti*o#ies inclu#e: <"
immune reaction #irecte# #estruction of neoplastic cells5 I" interference ,ith the gro,th an#
#ifferentiation of malignant cells5 %" antigen epitope #irecte# transport of anti3cancer agents to
neoplastic cells5 @" anti3i#iotype tumour .accines5 an# L" #e.elopment of engineere#
humani-e#" mouse m(*s for anticancer therapy. 'n a##ition0 a .ariety of agents e.g. to1ins0
ra#ionucli#es0 chemotherapeutic #rugs" ha.e *een conjugate# to mouse an# human m(*s for
selecti.e #eli.ery to neoplastic cells.
UNIT 7I CU--ENT TO+ICS IN IMMUNOLOGY
Summa$y o* a## "ection" !i"cu""e! a%o'e