Hybridoma technology allows for the production of monoclonal antibodies through the fusion of B cells and myeloma cells. Georges Kohler and Cesar Milstein discovered this technique in 1975. Mouse spleen cells producing antibodies targeting a specific antigen are fused with myeloma cells. The resulting hybridoma cells can be cloned, grown indefinitely in culture, and produce large quantities of identical monoclonal antibodies directed against a single epitope of the target antigen. Monoclonal antibodies find applications in areas such as disease diagnosis, pregnancy testing, and blood typing.

1. HYBRIDOMA TECHNOLOGY
Production of monoclonal antibodies and their
applications
By
Breena

2. INTRODUCTION
 Hybridomas are cells which are have been engineered to generate a wished
antibody in huge amount
 To generate monoclonal antibodies, B-cells are taken from the spleen of
animals and they are fused with myeloma tumor cells which grow
indefinitely in culture
 Monoclonal antibodies are generated in unique cells through a method
which is called as hybridoma technology
 In the year of 1975, two scientists discovered hybridoma technology and
they were Georges Kohler of West Germany and Cesar Milstein of
Argentina.

3. METHODOLOGY
 Hybridoma is produced by the antibody-producing cell obtained from the
mouse's spleen. The specific antigen is injected into a mouse, procuring the
antigen-specific plasma cells. Then the fusion of this cell takes place with a
cancerous immune cell called a myeloma cell
 The hybrid cell, which is thus produced, can be cloned to produce many
identical daughter clones. These daughter clones then secrete the immune
cell product
 These antibodies come from only one type of cell (the hybridoma cell) they
are called monoclonal antibodies
 Benefits of these cells are mentioned below:
 It has ability to combine two distinct types of cells
 Ability to grow continually
 Ability to generate pure antibodies in huge amount

4. CONTINUE…

5. EXPLANATION
 Laboratory animals e.g. (mice) are first exposed to an antigen to which we
are interested in isolating an antibody against
 Splenocytes are isolated and the B cells are fused with myeloma cells
lacking HGPRT(hypoxanthineguanine phosphoribosyltransferase) gene -
using polyethyleneglycol

6. HAT MEDIUM
 Incubation of fuse cells is done in HAT Medium
 HAT Medium is (hypoxanthine-aminopterin-thymidine medium) is a
selection medium for mammalian cell culture, which relies on the
combination of
 Aminopterin , a drug that acts as a powerful folate metabolism inhibitor
 The trick is that aminopterin blocks DNA denovo synthesis, which is
absolutely required for cell division to proceed, but hypoxanthine and
thymidine provide cells with the raw material to evade the blockage (the
"salvage pathway"), provided that they have the right enzymes, which
means having functioning copies of the genes that encode them

7. CONTINUE…
 Aminopterin which is present in myeloma cells die as they cannot generate
nucleotides by de novo or salvage medium prevents the block way which
permits for nucleotide synthesis so B cells and D cells which are not fused
die as they have a short lifespan
 B cell myeloma hybrids do not die, they survive as HGPRT gene which is
coming from B cells is functional
 These cells generate antibodies which are immortal. After this, an incubated
medium will be diluted into multiwell plates, as antibodies in a B cell are
generated by similar B cell
 Because they are generated by same B cell they get directed towards the
same epitope so they are known as monoclonal antibodies
 Once a hybridoma colony is established, it will continuallygrow in culture
medium like RPMI-1640 and produce antibody

8. CONTINUE…

9. CONTINUE…
 The next stage is a rapid primary screening process, which identifies and
selects only those hybridomas that produce antibodies of appropriate
specificity
 Multiwell plates are used initially to grow the hybridomas and after
selection, are changed to larger tissue culture flasks
 The culture supernatant can yield 1to 60 ug/ml of monoclonal antibody,
which is maintained at 20°C or lower until required

10. PURIFICATION OF ANTIBODIES
Antibodies can be purified by anyone of the following techniques
 Ion-exchange chromatography
 Antigen affinity chromatography

11. APPLICATIONS OF MONOCLONAL ANTIBODIES
1st monoclonal antibody was approved in 1986 for kidney transplant
rejection. In 1990 chimeric monoclonal antibodies was develop(2/3 of the
molecule contain human protein, rather than mouse protein) so these are
less rejected
 monoclonal antibodies are used to measure the level of hormone in the
blood
 Monoclonal antibodies are used to locate or identify specific moleculesin a
cell or tissue
 Various antibody preparations have been developed which facilitate the
imaging of vascular related conditions. For examples Myocardial infarction,
Deep vein thrombosis
 Monoclonal antibodies are used in pregnancy testingto detect a specific
hormone
 Monoclonal antibodies are used , for the identification of ABO blood groups
 Due to the presence of desired immunity, monoclonal antibodies are used in
the diagnosis of diseases