5. Hybridoma colony and antibody creation:
A particular antigen is infused into mouse. Myeloma, a harmful cell is intermixed with specific plasma cells.
This hybrid cell is therefore copied. Numerous specific daughter clones are delivered in immune cells. Antibodies are created from just a single kind of cell. At that point, antibody is created in HAT medium . Specific antigen is exposed to mice. splenocytes
are isolated from mouse and B cells are fused with immortal myeloma cells and HGPRT gene is absent. Polyethylene glycol helps in combination. Fused cells in the HAT medium are incubated. The instrument relies upon biosynthesis of nucleotides guarantees that just fused hybrids will survive. Tetrahydrofolate is importanr in nucleotide synthesis and this can be obtained by dihydrofolate reductase. Folic acid analouge aminopterin obstructs this catalyst. In the medium,consolidating 6-thioguanine or 8-azaguanine into nucleotides by HGPRT cause cells death. The cells where active HGPRT is missing can survive.
Along these lines, just survival partitions are B cell-myeloma hybrids. At that point, incubated medium is diluted into multi well plates are done as such that just a single cell can be available in each well and checking for specific antibody is occurred. (Tokunaga, Chiba and Ohnishi, 2010)(Kulkarni, 2002)