Immunological

techniques
Bio233
Fall 2009
Today’s agenda
Green sheet, manual
introduction.
Check in- lockers etc- Tim
Introduction to Bio 233
Animal handling –intro- Larry
Exercise #2-
◦ Collecting initial serum sample from
mice/freeze
◦ Assign hybridoma
Lab 1 - agenda and tentative
time line
Bio 233- Class hours
◦ M & W 9:30-2pm
◦ Several activities will require
additional outside hours
General information
Instructor- Tzvia Abramson Ph.D
◦ tzvia.abramson@sjsu.edu
◦ Office- DH 446 ext 44872
◦ Office hours: M & W 2:30-4:30pm
 Additional reading material and
PPT presentations will posted:
https://ewok.biology.sjsu.edu/DrAbr

Userid: ewok\biostudents
Password: 4biolecture
Assisting Bio233:
- Tim Andriese- Laboratory
services ext 44858
- Larry Young, Nelia- animal care
facility ext 44929.
 Bio 233
Practical lab Journal club
Lecture Notebook
work Students

Animal work Protein/Ab analysis Tissue culture

IACUC purification counting

Immunization
characterization Thaw/freeze
injection

Electrophoresis/
Blood collection Hybridoma
Western

Ascites production/
FACS
harvest

Spleen removal
Screening/ELISA
feeders
Prerequisites

◦ Biology 6 and biology 107 with a

grade >B or equivalent

◦ Summer Immunology workshop

◦ Please fill form with contact prereq

and goals in the manual
Books and references:
Required: Boothby: Immunological
Techniques Laboratory Syllabus -
Biological Students Association (DH
346) updated 2008
 
Recommended reading:
◦ Peter Parham - The Immune System, Third
Edition Garland Science Publishing 2009
◦ Harlow & Lane: Antibodies
(reference/recommended) Cold Springs
Harbor Press 1988
◦ Howard C Gary and Matthew R Kaser.
Making and using antibodies- a practical
handbook by Gary C. CRC 2007
Grading
Grades
◦ 30%- Exam 1 -midterm
◦ 30% -Exam 2 -final
◦ 15%- Research paper (10%
presentation, 5%
evaluation by class)
◦ 15%- Laboratory notebook (5% x 3)
◦ 10%- Evaluation by instructor

No late examination without prior

permission from instructor.
Instructors evaluation
10%
Collegial and polite work propriety
◦ Partner, rest of the class including
assisting personnel.

Arriveprepared knowing the

material

Apply safety rules.

Work neatly, leave the work station

properly.
Notebook (pg 3-4)
 Keep laboratory notebook divided in two sections that
contain:
◦ Table of contents
◦ Numbered pages
◦ The following information for each experiment:
Title- Name of experiment corresponding to table
of context.
Background / introduction
Material and methods- briefly state the
experiment, deviation from original protocol,
reference syllabus
Results- presentation of data, figures, tables
Safety, Safety, Safety!
Read and sign safety lab paper!
Closed shoes
No shorts
Lab coat at all times
Tie up long hair
Gloves discarded in biohazard
trash.
Discard lab equipment properly
Control emotions- communicate
with instructor.
LATE OR RETROACTIVE
ADDS:
Itis the student's responsibility
to determine that they are
officially enrolled in courses as
determined on 'My SJSU' or by
confirming with the course
instructor BEFORE the last day to
add.

No late or retroactive add forms

will be approved in the
 BIOLOGY/CHEMISTRY 233
IMMUNOLOGICAL TECHNIQUES

BSA
DH346

5$

JOHN BOOTHBY, PH.D.

Instructor : Tzvia Abramson Ph.D
DEPARTMENT OF BIOLOGICAL SCIENCES
& DEPARTMENT OF CHEMISTRY
SAN JOSE STATE UNIVERSITY
FALL2009
Fall 2005
MANUAL PURCHASE

LAB CHECK-IN/ TIM:

EQUIPMENT
PIPETTES
MICROSCOPES
HALLWAY CABINETS
LAB-COATS
Overview-
Monoclonal Antibodies production
Host Defenses
First line- PHYSICAL
BARRIERS
Second line- Innate immune
system
Third line-
THE ADAPTIVE IMMUNE RESPONSES TO INFECTION
MATURE B CELLS ENCOUNTERING ANTIGEN IN
LN FORM GERMINAL CENTERS AND MATURE
INTO PLASMA CELLS
Tortura et al. 2006 Figure 17.7
T cell receptor and B cell receptor
provide the specific immune response
Antibody structure
Antibody structure
X-ray crystallographic structure of IgG
Antibodies- antigen
binding
Epitope recognition by variable
regions of heavy and light chains
Hyper variability of V domain
in both heavy and light
chains
Generating diversity in B cell receptor/
antibodies-
Gene rearrangement, class switch and somatic
mutation
Antibody isotypes
Antibodies Isotypes and
function
 ONE B CELL- ONE TYPE OF
ANTIBODY

www.healthsystem.virginia.edu/
 Humoral immunity-
Antigenic Determinants

 Antibodies recognize and react with antigenic

determinants or epitopes on an antigen.

Tortura et al. 2006 Figure 17.1

Polyclonal/monoclonal antibodies
Köhler and Milstein –Nobel prize
medicine 1984
monoclonal antibody production
 Why bother generate
monoclonal antibodies?
 Polyclonal Ab
◦ Detects a multiplicity of epitops on an antigen.
◦ Simple and cheap to produce
◦ Difficult to reproduce exact same Abs.

 Monoclonal Abs
◦ Detects a single epitope on an antigen
◦ Continuous culture of B cell hybridoma offers
reproducibility
◦ Offer powerful reproducible tools for research
clinical diagnostics and clinical treatment
(humanized Abs)
Monoclonal antibody
production
Monoclonal antibody
production
HOW DO WE MAKE MONOCLONAL ANTIBODIES?

 Immunization
(polyclonal
antibodies)
 Harvest B
lymphocyte (spleen
or lymph nodes)
 Fusion of B cells
with immortal cells
 Selection for the
preferred clones
 Characterization
and
further developments
Immunization

4 times mice
immunization with
2 weeks intervals
Fusion and screening for
desirable hybridoma
Expansion of monoclonal
antibody

In vitro

In vivo- ascites

production
 Characterization of Abs
 Isotype determination

 Purification

 Specificity.
APPLICATION OF mAbs

Research / Clinical:
diagnostics: Diagnostic-
Protein functional cell population
analysis- in peripheral
blocking/neutralizin blood.
g, detection in
Western blots. Treatments

Cellsurface
identification- CD-
Flow cytometry- identify and quantify cell
subsets
Confocal / fluorescent microscopy-
visualize cells and organels
THERAPEUTIC MONOCLONAL ANTIBODIES

CD3-block CD20 Rituximab- Infliximab a4 TNFα

T cell mediated B cell- depletion by homing receptor Humira
responses that ADCC NK. Multiple sclerosis RA
reject transplanted non-Hodgkin
kidneys B cell lymphoma
Carter Nature Reviews Immunology 6, 343–357 (May 2006) | doi:10.1038/nri1837
Monoclonal Abs production
 mAb production in Bio 233

II
Production of Listeria specific hybridoma cell

I
Section #1 (page 1)
________________________________________________________________________
BIOLOGY/CHEMISTRY 233
IMMUNOLOGICAL TECHNIQUES
TABLE OF CONTENTS
Production specific monoclonal antibody-producing hybridomas:
Preparation of antigen ----------------------------------------------------------------------------

Immune responses to antigens in BALB/C mice -------------------------------------------

Cell culture records (SP2/0)----------------------------------------------------------------------

Fusion, selection and culture---------------------------------------------------------------------

Screening for specific antibody producing cells --------------------------------------------

Cloning/screening for monoclonal antibody-producing hybridomas ----------------

________________________________________________________________________
Expansion of mAb in- vivo and in-vitro
and characterization

I
I
________________________________________________________________________

Section #2 (page 75)

________________________________________________________________________
BIOLOGY/CHEMISTRY 233
IMMUNOLOGICAL TECHNIQUES
TABLE OF CONTENTS
Production and characterization of monoclonal antibodies:
Production of monoclonal antibodies in ascites in BALB/C mice ---------------------

Production of monoclonal antibodies in cell culture---------------------------------------

Purification of antibody from supernatant/ascites ----------------------------------------

Quantification of purified antibody from supernatant/ascites --------------------------

Determination of monoclonal antibody specificity by ELISA ---------------------------

Determination of monoclonal isotype by flow cytometry -------------------------------

Determination of monoclonal antibody purity by SDS-PAGE --------------------------

Titration of purified antibody by ELISA -----------------------------------------------------

Test purified antibody in immunoblot application-----------------------------------------

________________________________________________________________________
 Bio 233 –lab schedule F-
2009
Lab D Hybridom Purification & Journal
# ate as characterizat Club
productio ion (JC)
n of mAb
 Laboratory
notebook
divided in part I and
II

II
 Today
I II
 Hybridoma production  Ab expansion and
Today purification-August 6
◦ Preimmune Prime mice with
serum/tail bleeding pristane for ascites
production (done)
◦ Assign hybridoma
Preimmune serum-tail bleeding
 Negative serum for
all screening tests.
 Leave collected blood
on bench for 10 min.
 Centrifuge 10min
 Transfer serum to a
new microcentrifuge
tube labeled and
refrigerate -20oc.
Next lab – Wednesday August
26

I II
 Hybridoma  Ab expansion and
production purification
◦ 1st immunization ◦ Inject hybridoma
cells for ascites
production in mice