Clinical Serology-Immunology Lecture

LABEL
IMMUNOASSAY
 LABELED IMMUNOASSAYS
 Ag and Ab in small concentrations
 needs labeled molecules for
quantitation

Indicator
labels
 ANALYTE
 substance to be measured
 bound by molecules that react
specifically to them
 
Constituents of Labeled
Immunoassays
Labeled
Detectio Analyte
n

Antibodi
es

Separati
on
Standards/
Calibrators
 LABELED ANALYTE

Radioactive Isotopes Fluorochromes Enzyme

Alkaline
absorb light phosphatase
125 I horseradish
peroxidase
Fluorescence
antigen:
spectrophotometer
131 I spectrofluorometer or
antibody:
flow cytometer
luminometer
 ANTIBODIES
 sensitivity depends on the affinity

 specificity of antigen to antibody is

also important
 STANDARDS OR CALIBRATORS

 unlabeled analytes

 to establish a relationship between

the labeled analyte
 SEPARATION

Centrifugation
Precipitation
or filtration

sandwich Solid-phase
technique separation
 DETECTION
 Presence of labeled analyte
 Immunoassays

Are based on

Immunoprecipitation Ab-Ag interaction Immunoassays

Which may be
Detected

using

Indicator Labels

Radioactive Enzymes Fluorochrome

Isotope
 LABELED IMMUNOASSAY

RADIO
IMMUNOASSAY
RADIOIMMUNOASSAY
(RIA)
Developed by Yalow and Berson

Direct binding assay

131 I, 3H, 125I

ADVANTAGES
Can measure hCG, FSH, gastrin, insulin, CEA,
thyroxine, TSH, estrogens, androgens, and IgE

Extremely sensitive and precise technique

Can determine small or trace amounts of analytes

that are small in size
DISADVANTAGES
Health hazard involved in working with radioactive substances

More difficult and expensive to maintain a laboratory license in

compliance with federal law

Disposal problems

Short shelf life

Expensive equipment
Competitive binding assay

PRINCIPLE INDICATOR PURPOSE

Labeled and To determine
unlabeled
LABEL
the presence
antigens can Radioactive
of antigen or
compete equally labels
for the same antibody in the
Ex. 125I biological
binding site on
the antibody sample
 Competitive binding assay
Radiolabeled
Known quantity of
antigen mixed with a
an antigen is made
known amount of
radioactive
antibody

t
w
o
c
h
e
m
ic
al
ly
bi
n
d
to
o
n
e
a
n
ot
h
er
unknown quantity of that
●

same antigen is added

unlabeled antigen compete with

●

the radiolabeled antigen for

antibody binding sites

unlabeled antigen will displace

●

the radiolabeled variant

 ratio of Ab - bound radioactivity of the
radiolabeled antigen bound antigens are free antigen
to free radiolabeled separated from the remaining in the
antigen will be unbound ones supernatant is
reduced measured
The amount of label
in the bound phase
is indirectly
proportional to the
amount of patient
antigen present.
As the amount of patient antigen increases, fewer binding sites will be
occupied by labeled antigen.
 IMMUNORADIOMETRIC ASSAY
(IRMA)
Noncompetitive Immunoradiometric Assay

the reversible and non-covalent

Uses labeled antibody that is present
binding of antigen by a specific
in excess
labeled antibody
The supernatant
●

containing the bound

complexes is counted

The amount of bound labeled

●

antibody is in direct proportion

to the amount of patient analyte
 Immunoradiometric Assay
(IRMA)
Advantage

Disadvantage
Faster reaction rate

Increased sensitivity

Loss of specificity
Excess antibody allows all of the unknown analyte to be involved in the reaction

Increase in antibody concentration can

result in cross reactivity with other antigens
 LABELED IMMUNOASSAY

ENZYME LABEL
IMMUNOASSAY
ENZYME LABEL IMMUNOASSAY
Naturally
Non isotopic
occurring =
Label CHEAP

Quali and
Quanti
Spectro

HOMO or
Table 1. Examples of Enzyme Immunoassay Table 2. Enzymes used in Enzyme
Borrelia burgdorferi (IgG and IgM) Immunoassay
Cytomegalovirus (IgG and IgM Ab) ENZYME SOURCE
Cytomegalovirus (Ag) Acetylcholine Electrophorous
Hepatitis A (total Ab) Esterase electicus
Hepatitis B:
Alkaline Escherichia coli
Anti-HBs
Phosphatase
Anti-HBc
Anti-HBc Beta- Escherichia coli
Anti-HBc (IgM) Galactosidas
HBs Ag e
HBe Ag Glucose Aspergillis niqer
Oxidase
Hepatitis delta Virus (total Ab)
G6PD Leuconostoc
Hepatitis non-A and non-B mesenteroides
HIV Ab
Lysozyme Egg white
HIV Ag
HTLV-I Ab Malate Pig Heart
dehydrogena
HTLV-II Ab
se
Human B lymphocytic virus Ab
Rubella Virus (IgG and IgM Ab) Peroxidase Horse Radish
Toxoplasma gondii (IgG and IgM Ab)
 HETERO

Separatio ELISA and

Capture
n Step assays
HETEROGENOUS
ENZYME
IMMUNOASSAY
 ELISA
COMPETITIVE

Competition Bet. Enzyme

Labeled Ag and unlabeled Insulin and
Ag on the Ab attached to
a solid phase estrogen
 COMPETITIVE

Competitive assay, high concentration of analyte

 COMPETITIVE

Competitive assay, low concentration of analyte

COMPETITIVE

Color 1/α to
amount of
analyte present
NON COMPETITIVE
CAPTURE ASSAYS
●
Antibody bound to a solid phase

●
Antigens are captured
●
Multiple epitopes

●
Enzyme-labeled antibody

●
Enzymatic activity directly proportional to the
amount of antigen present
 CAPTURE ASSAYS
Solid-phase Patient’s
antibody antigen

Incubate

Enzyme-
labeled
Colored
antibody reaction

Spectrophot
 CAPTURE ASSAYS
Antigens

Antibodies

Polypeptide hormones

Proteins

Tumor markers

Microorganisms
CAPTURE ASSAYS
 CAPTURE ASSAYS

Remember:

●
Capture antibody
●
High specificity
●
High affinity
MEMBRANE-BASED CASSETTE
ASSAYS
Qualitative > quantitative

Rapid and easy to perform

Gives instant results

Home and point-of-care testing

Single use

Disposable plastic cartridge

MEMBRANE-BASED CASSETTE ASSAYS
MEMBRANE-BASED CASSETTE ASSAYS

Nitrocellulose membrane

Immobilize Immobilize
proteins nucleic acids
MEMBRANE-BASED CASSETTE ASSAYS

Separate type

●
Separate addition of:
●
Patient’s sample
●
Wash reagent
●
Labeled antigen or antibody
●
Substrate
MEMBRANE-BASED CASSETTE ASSAYS

Combined type/
Immunochromatography

●
All requirements are combined
already in the cassette
MEMBRANE-BASED CASSETTE ASSAYS

●
Labeled zone
●
Analyte combines with the
labeled antigen or antibody

●
Detection zone
●
Captures the immune complex
●
Formation of colored line or a plus
sign (+)
MEMBRANE-BASED CASSETTE ASSAYS
MEMBRANE-BASED CASSETTE ASSAYS

Microorganisms
Streptococcus

Pregnancy testing
Hepatitis
Heart attack
Troponin
Virus
Rubella
HOMOGENOUS
ENZYME
IMMUNOASSAY
 Antigen-antibody system in which no
separation step is needed

Less sensitive than Heterogeneous assay

No washing step necessary

Major use
Determination of low molecular weight analytes

Horomones Threpeutic drugs Drugs of abuse

Principle
 Change in enzyme activity as specific
antigen-antibody combination occurs

Antigen labeled Antibody binds to Enzyme active site

with enzyme tag determinant blocked
 Competitive assay

proportio
nal
 Detectability
(enzyme

binding
activity)

(antibody)

Strength of
sensitivity

(interference)
Susceptibility
activity
Change in
INTERFERENCES:
a. Endogenous enzyme activity
b. Cross reacting antigens
c. Enzyme inhibitors
CLONED ENZYME DONOR
IMMUNOASSAY
GENETIC ENGINEERING

β-GALACTOSIDASE

TWO SUBUNITS
Subunits
A. Large polypeptide- enzyme acceptor
B. Smaller subunit- enzyme donor
• Small piece- attached as label to antigen

• The complex will compete with patients

antigen
 Enzyme
Labeled
Ab Activity
Ag
reduced
 Enzyme
Patient
Ab activity
Ag
greater
ADVANTAGES AND DISADVANTAGES OF
ENZYME IMMUNOASSAY

ADVANTAGE DISADVANTAGE
High sensitivity Inhibitors

Cheap instrumentation Size of enzyme label

Cheap and long lasting Non specific protein

reagents binding
Requires no Sensitivity of enzyme to
separation(homogeneous) temperature
 LABELED IMMUNOASSAY

FLOURESCENCE
IMMUNOASSAY
• ALBERT COONS –
developed the fluorescent
method of labeling proteins, a
significant tool for the study of
infection in human beings.

• Antibodies could be labeled

with molecules that fluoresce
called Fluorochrome /
Fluorophores
•sensitive technique

• measurement of many compounds,

including drugs, hormones, and proteins;

•Identification of antibodies

• Quantification of antigens
 Fluorochrome or
Fluorophores

FLUORESCEI
RHODAMINE
N

ISOTHIOCYANATES
FLUORESCEIN ISOTHIOCYANATE

Absorbs maximally at 490 – 495

GREEN color at 517 nm

Highly sensitive

Good photostability
TETRAMETHYLRHODAMINE

Absorbs at 550 nm

RED light at 580 – 585 nm

 TAMR USED
FITC A
TOGETH
ER
PHYCOBILIPROTEIN

Newer compound used

Emits red flourescence

At over 600 nm
FLUORESCENCE MICROSCOPY

LIGHT SOURCE – EMITS

LIGHT IN THE APPROPRIATE
WAVELENGTH TO EXCITE
THE FLUOROCHROME

HIGH INTENSITY LIGHT

SOURCES SUCH AS:
TUNGSTEN HALOGEN
MERCURY VAPOR ARC
FI
LT ●
Between light source and

E FIRST ●
specimen
Remove uneccessary
wavelengths
R
S
SECO ●
Between specimen and ocular
lens
Screens out light other than that
ND
●

produced by the fluorchrom

 FLUORESCENT
STAINING

Antigen Ag and Ab
Detection Detection
DIRECT IMMUNOFLUORESCENT ASSAY
Ab conjugated Added directly
with fluorescent to Ag fixed on
tag slide

Incubate
and wash

Ag appear BRIGHT
Viewed using APPLE GREEN or
Fluorescence ORANGE-YELLOW
OBJECTS against dark
Microscope background
DIRECT IMMUNOFLUORESCENT ASSAY
Best suited for Antigen
Detection of:

Legionella pneumophilia

Pneumocystitis carinii
DIRECT IMMUNOFLUORESCENT ASSAY
Best suited for Antigen
Detection of:

Chlamydia trachomatis

Respiratory Syncytial virus

INDIRECT IMMUNOFLURESCENT ASSAY
Wash and an Anti-
Patient Serum Human Immunoglobulin
containing fluorescent
+ Known Ag tag is added

Fluorescence
is determined

Amount of fluorescence
is directly proportional
to amount of patient
antibody present
DIRECT IMMUNOFLURESCENT ASS
Useful in antibody
detection of

Treponema
Antinuclear
Chlamydia
Toxoplasma
Herpes simplex virus
Epstein – Barr virus
Cytomegalo virus
 HETEROGENOUS FLUORESCENT
IMMUNOASSAY
• Require a Separation Step

•Includes :
• Indirect Assays
• Competitive Assays
• Sandwich or Capture Assays

•Based on the principles of Enzyme

Immunoassay but the label is Fluorescent that
can be applied to either Antigen or Antibody.
 HETEROGENOUS FLUORESCENT
IMMUNOASSAY
• Used to Detect compounds such as:
• Cortisol
• Progesterone
• Serum Thyroxine (T4)

• Solid Phase Fluorescent Assays identifies:

• Ab to Nuclear Ag
• Toxoplasma Ag
• Rubella virus
• Other virus Ags
 SOLID PHASE SEPARATION
ds
o b ea
i c r
Microbeads are
Ag or Ab
m used attaches to the
beads

React with analyte

and a fluorescent
labeled analyte

Mixture is
centrifuged; Analyzed for
supernantant fluorescence
discarded
 SOLID PHASE SEPARATION
t i ck
i p s Coated with
DDipstick Ag or Ab

Reacted with
Patient Sample

One side of the

Labeled Ab is stick is not coated
then added (serves as control)
 HOMOGENEOUS ASSAYS
No Separation step ; Only one incubation step and no wash step

Basis: Change that occurs in the Fluorescent label on Ag when it binds to specific Ab

Such changes may be related to: Wavelength emission, Rotation freedom, Polarity or
Dielectric strength

Amount of fluorescence is directly proportional to amount of Antigen

Not Sensitive
 FLUORESCENCE POLARIZATION
IMMUNOASSAY
n change
esent, Less
Ab is bound
ation ofis
arization
ent light

De
r
e
f
o
ar
za
o

s
nv
r
el

r
p
rt
o
a
to
o
c
n
a
o

f
n
ly
e
IF LABELED MOLECULE IS BOUND TO AN ANTIBODY, THE
MOLECULE IS UNABLE TO TUMBLE AS RAPIDLY AND IT
EMITS AN INCREASED AMOUNT OF POLARIZED LIGHT,

DEGREE OF POLARIZED LIGHT REFLECTS AMOUNT OF

LABELED ANALYTE THAT IS BOUND
FLUORESCENT IMMUNOASSAYS

Advanta ●
More sensitive than radiolabels
and enzyme reactions

ges Simple and no hazardous wastes

●

Disadva ●

●
Non specific binding can cause change in
fluorescence
Bilirubin or Hemoglobin present can absorb

ntages
either excitation or emission energy
●
Expensive
 LABELED IMMUNOASSAY

CHEMILUMINESCEN
CE IMMUNOASSAY
Chemiluminescent Immunoassay

Chemiluminescence
- emission of light caused by a chemical
reaction
- produces an excited molecule that decays
back to its original ground state - measured
using a luminometer.
Common Chemiluminescent substances

Luminal

Acridium esters

Peroxyoxalates

Ruthenium
derivatives

dioxetanes
 Chemiluminescent Immunoassay

Oxidized
Chemiluminescen
(hydrogen peroxide +
t substances
enzyme)

In
te
r
m
e
di
at
e
s
(h
ig
h
er
e
n
er
g
y
st
at
e)
Chemiluminescent Immunoassay

Heterogenous Homogeneous
assays assays

*competitive assay

* Sandwich format

** labels can be attached either to the

antigen or antibody**
Chemiluminescent Immunoassay

Advantages
Reagents are
Have excellent
stable and
sensitivity relatively non-toxic
 AREVALO-GALANG-CERVANTES-CABANAG-CRUZ-CARLA-KHAN-
BERNARDES-MARIANO-BAUTISTA-ZABALLERO-MOLANO
3HMT

CLINICAL SEROLOGY AND IMMUNOLOGY LECTURE

LABELED
IMMUNOASSAY
Assoc. Prof. Jennifer Tiburcio