hYBRIDOMA TECHNOLOGY.pptx

hybridoma

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

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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 A minopterin   , 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

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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 1 st 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