Monoclonal antibodies are been developed an produced from some identical parental immune cells. they can be developed to target and identify specific cells and antigens and to work as antibodies in tandem with the human immune system against them. Hybridoma technology was developed by Georges J.F. Kohler and Cesar Milstein. they made a hybrid cell that will make number of monoclonal antibodies against them.

1. PRODUCTIONOFMONOCLONAL
ANTIBOBYBYHYBRIDOMA
TECHNOLOGY
MADHUMA K

2. MONOCLONAL ANTIBODY
Monoclonal antibodies are
antibodies that been developed
and produced from some
identical parent immune cell.
 They can be developed by
scientists to target and identify
specific cells and antigens and to
work as antibodies in tandem
with the human immune system
against them.

3. APPLICATIONS
 DIAGANOSTIC TESTING
PREGNENCY TESTING
RADIO IMMUNO DETECTION OF CANCER
TREATMENT OF CANCER THROUGH DRUGS
VIRAL DISEASE TREATMENT
IDENTIFING PATHOGENS.
TRACING SPECIFIC CELLS AND THEIR FUNCTION

4. HYBRIDOMA TECHNOLOGY

5. HYBRIDOMA TECHNOLOGY
This technology was developed
by Georges J.F. Kohler and Cesar
Milstein.
 in 1984 they shared a Nobel
prize for this discovery.
They make a hybrid cell that will
make a number of monoclonal
antibodies against antigen.

6. PROCEDURE
1. Immunization of specific animal which generate
hybridoma cell with spleen cell.
2. Isolation of myeloma cells
3. Fusion between spleen cell and myeloma cell
4. Selection of HAT medium
5. Isolation of hybridoma cell
6. Screening of hybridoma cells.

7. 1.IMMUNIATION OF SPECIFIC ANIMAL
 An antigen immunized to an
animal like mice via
intravenously ( directly to
blood ) by injection.
Where in spleen it activates
b-cell which produce plasma
cell (spleen cells).
spleen cell is isolated from
the spleen of the animal.

8. 2.ISOLATION OF MYELOMA CELLS.
Myeloma cells are cancerous cells which is
isolated from bone marrow.
Myeloma cells are generally immortal in
nature ( that which never dies) and has
multiplication property.

9. 3. FUSION OF SPLEEN CELL AND MYELOMA CELLS
 It requires PEG ( poly ethylene glycone) medium
for fusion.
It can also done by electro fusion
Fusion between spleen cell and myeloma cells
produce 5 different types of cells.
•Fused plasma
•Fused myeloma
•Hybridoma
•Unfused plasma
•Unfused myeloma

10. 4.SELECTION OF HAT MEDIUM
(Hypoxanthine, Aminopterin, Thymidine)
 the hybridoma cells or fused cells are selected by
incubated in the selective media called HAT medium.
It contain hypoxanthine , aminopterin and thymidine.
The unfused b cell have a very short life span in the
medium.
The myeloma cells can synthesis DNA nucleotide using
two pathway : Denovo pathway and Salvage pathway.
In HAT medium myeloma cells cannot replicate
because denovo pathway is blocked by the
aminopterin in the medium.

11. SELECTION IN HAT MEDIUM
when denovo pathway is blocked cell will
utilize alternate pathway called salvage
pathway. But it cannot take place due to
the lack of HGPRT ( hypoxanthine
guanine phosphoribosyl transferase).
 so it is contributed by B cells which is
rich in HGPRT+
But the salvage pathway is also inhibited
due to the mutation of enzyme thymidine
kinase ( TK) an enzyme that catalyse the
phosphorylation reaction.

13. 5. ISOLATION OF HYBRIDOMA CELLS
 Fused myeloma and unfused myeloma cannot
survive in HAT medium because of the absence
of HGPRT enzyme.
Fused plasma and unfused plasma cells have
HGPRT enzyme but they have only very short life
span.
 But hybrid cells has HGPRT enzyme from the
spleen or plasma cells as well as they have the
ability to multiply repeatedly as the myeloma
cells.
So isolation of hybrid cell is possible because
they are the only cell that survive in the HAT
medium.

14. 6. SCREENING OF HYBRIDOMA CELLS
 ELISA screening method is used for the
screening of the hybridoma cells
Hybridoma cells are multiplied using in
vivo or in vitro method.
In in-vivo procedure hybridoma cell is
introduce in to the peritoneal cavity of
animal.
In-vitro method involves culturing of
hybridoma cells in suitable cultural
media an then antibodies are isolated
and purified.