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5. Hybridoma colony and antibody production:
A specific antigen is injected into mouse. Mouse spleen produces the antigen-specific plasma cells and myeloma, a cancerous cell is intermixed with these cells. This hybrid cell is thus duplicated and many identical daughter clones are produced and then secretion results in immune cells. Thus monoclonal antibodies are produced from only one type of cell. Then, monoclonal antibody is produced in HAT medium (hypoxanthine aminopterin thymidine). To obtain antibody against a specific antigen, that antigen is exposed to mice. Segregation of splenocytes from the mammal and fusion of the B cells with immortal myeloma cells that lack HGPRT gene is occurred. Polyethylene glycol or Sendai virus aids in fusion. Incubation of intermixed (fused) cells in the HAT medium occur. The mechanism depends on biosynthesis of nucleotides assures that only fused hybrids will survive. Tetrahydrofolate is essential for nucleotide synthesis and this can be acquired by dihydrofolate reductase. Folic acid analogue aminopterin blocks this enzyme. Incorporating 6-thioguanine or 8-azaguanine into nucleotides in the medium by HGPRT cause the death of cells. The cells where active HGPRT is absent can survive. So, only survival portions are B cell-myeloma hybrids. Thus, antibodies production by these cells (characteristics of B cells) and cell`s immortality (characteristics of myeloma cells) happen. Then, dilution of incubated medium into multi well plates are done so that only one cell can be present in each well and checking for desired antibody is taken place. (Tokunaga, Chiba and Ohnishi, 2010)(Kulkarni, 2002)

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