Hybridoma technology and application for monoclonal antibodies

very nice and easy language..

1. Presented by:-
Jagohool Singh
Roll no=1237
MSc.Biotechnology 1st
MDU ROHTAK

2. 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.
Polyclonal antibodies are antibodies that are
derived from different cell lines.

3. 1975, Georges Köhler and Cesar Milstein
- awarded Nobel Prize in1984

4. In 1975, Kohler and Milstein first
fused lymphocytes 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.

5. 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)

6. PRODUCTION OF MONOCLONAL ANTIBODY
Step 2: - Preparation of Myeloma Cells
HYBRIDOMA TECHNOLOGY
Myeloma Cells
HGPRT
-
(Hypoxanthin Guanine
phosphoribosyltransferase)

7. PRODUCTION OF MONOCLONAL ANTIBODY
Step 3: - Fusion of Myeloma Cells with Immune Spleen Cells
&
Selection of Hybridoma Cells
HYBRIDOMA TECHNOLOGY
FUSION
PEG
MYELOMA CELLS
SPLEEN CELLS
HYBRIDOMA CELLS
ELISA PLATE
Feeder Cells
Growth Medium
HAT Medium
1.Plating of Cells in
HAT selective
Medium
2.Scanning of Viable
Hybridomas

11. Selected by using HAT medium (Hypoxanthine-
Aminopterin-Thymidine)
Myeloma cells are unable to grow
B cells are able to survive, but can not live for extended
periods

13. (4) Fusion of myeloma and B cells (using PEG)
(5) Separation of cell lines

14. (6) Screening of suitable cell lines

15. (7) in vitro (a) or in vivo (b) multiplication
(8) Harvesting

17.  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.

18. 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.

19. The test of HIV
infection is based on
detecting the
presence of HIV
antibody in the
patient’s blood
serum.

20. 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.

21. 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.