monoclonal antibodies

1. Hybridoma technology
and applications of
monoclonal antibodies
Sri Ranjani Tallam
Dept of Zoology
D.K.Govt (A) college for Women
Nellore.

2. What are antibodies….
An antibody is a protein used by the immune system to identify and
neutralize foreign objects like bacteria and viruses. Each antibody
recognizes a specific antigen unique to its target.
Monoclonal antibodies (mAb) are antibodies that are identical because
they were produced by one type of immune cell, all clones of a single
parent cell. ( Clone is a group of genetically identical cells.)
Polyclonal antibodies are antibodies that are derived from different cell
lines.

3. Differences between monoclonal and polyclonal
antibodies

4. Hybridoma Technology
1975, Georges Köhler and Cesar Milstein
- awarded Nobel Prize in1984

5. In 1975, Kohler and Milstein first
fused lymphocytes with myeloma cells to
produce a cell line which was both
immortal and a producer of specific
antibodies. The two scientists were
awarded the Nobel Prize for Medicine in
1984 for the development of this
"hybridoma." The value of hybridomas to
the field was not truly appreciated until
about 1987, when MAbs were regularly
produced in rodents for diagnostics.

7. HYBRIDOMA TECHNOLOGY

8. STEPS :
1.Immunization
2. Cell fusion
3. Selection of hybridomas
4. Screening the products
5. Cloning and propagation
6. Characterization and storage

9. PRODUCTION OF MONOCLONAL ANTIBODY
Step 1: - Immunization Of Mice & Selection Of Mouse
Donor For Generation Of Hybridoma cells
HYBRIDOMA TECHNOLOGY
ANTIGEN ( Intact cell/
Whole cell membrane/
micro-organisms ) +
ADJUVANT
(emulsification)
Ab titre reached in Serum
Spleen removed
(source of cells)

11. PRODUCTION OF MONOCLONAL ANTIBODY
Step 3: - Preparation of Myeloma cells with defective
HGPRT Gene .
HYBRIDOMA TECHNOLOGY
Myeloma Cells
HGPRT-
(Hypoxanthin Guanine
phosphoribosyltransferase)

12. PRODUCTION OF MONOCLONAL ANTIBODY
Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells
&
Selection of Hybridoma Cells
HYBRIDOMA TECHNOLOGY
FUSION
PEG
MYELOMA CELLSSPLEEN CELLS
HYBRIDOMA CELLS
ELISA PLATE
Feeder Cells
Growth Medium
HAT Medium
1. Plating of Cells in
HAT selective
Medium
2. Scanning of Viable
Hybridomas

17. • Mammalian cells can synthesize nucleotides by two different
pathways:
• 1. Denovo Pathway :Aminopterin blockes the DNA synthesis
• 2. Salvage Pathway: The enzymes catalyzing the salvage pathway
include hypoxanthine-guanine phosphoribosyl tranferase (HGPRT).
• Myeloma cells lack HGPRT. That’s why can not synthesize the
nucleotides.
• HAT is the selective medium which contains Hypoxanthin,
Aminopterin and Thymidine.

18. Pathways for nucleotide synthesis

23. Application Of MAbs:
 Monoclonal antibodies are proving to be
very useful as diagnostic, imaging, and
therapuetic reagents in clinical medicine.
Many monoclonal antibody diagnostic
reagents now available are products for
detecting pregnancy, diagnosing
numerous pathogenic microorganisms,
measuring blood levels of various drugs,
and detecting antigens shed by certain
tumors.

24. Monoclonal – Diagnostic use
 A monoclonal antibody can be used to detect pregnancy only 14 days after conception. Other
monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal, and
Chlamydia infections.
They can be used to detect for the presence and quantity of this substance, for instance in a
Western blot test (to detect a substance in a solution) or an immunofluorescence test.
Monoclonal antibodies can also be used to purify a substance with techniques called
immunoprecipitation and affinity chromatography.

25. Pregnancy Tests
 A pregnant woman has the hormone human chorionic
gonadotrophin (HCG) in her urine.
 Monoclonal antibodies to HCG have been produced. These have
been attached to enzymes which can later interact with a dye
molecule and produce a colour change.

26. HIV DETECTION
a)HIV antigen is attached to the plate.
b)Patients serum passed over the plate. Any HIV
antibody in the patients serum will attached to the
antigen already on the plate.
c)A second antibody which is specific to the HIV
antibody is passed over the plate. This antibody will
attach to the concentrated HIV antibody on the
plate. This second antibody has an enzyme attached
to its structure.
d)Chromagen dye is passed over the complex of
concentrated HIV antibody/conjugated antibody.
e)The enzyme will turn the chromagen to a more
intense colour. The more intense the colour, the
greater the HIV antibody level. This would be the a
positive result for a HIV test.

27. Treatment of Cancer
 Cancer cells carry specific tumour-associated antigens (TAA) on
their plasma membrane.
 Monoclonal anti-TAA antibodies have been produced.
 Drugs which kill tumour cells or inhibit key proteins in tumour cells are
attached to monoclonal anti-TAA antibodies.
 Cancer cells are specifically targeted, avoiding damage to healthy
host cells.

29. Any questions ?
Thank you !!!