HAT medium

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HAT Medium (Hypoxanthine Aminopterin Thymidine ) is a medium used for preparation of monoclonal antibodies. This process is called Hybridoma technology. In this method, B cells are fused with HGPRT (Hypoxanthine Guanine Phosphoribosyl Transferase) negative myeloma cells (which have immortal property) using polyethylene glycol or the Sendai virus.
Humans can produce nucleotides via 2 pathways: the de novo pathway or salvage pathway. HGPRT and thymidine kinase are important enzymes for the salvage pathway, and therefore HGPRT negative myeloma cells cannot synthesize nucleotides via salvage pathway. Fused cells are incubated in the HAT medium. Aminopterin in the medium blocks the de novo pathway. Hence, unfused myeloma cells die, as they cannot produce nucleotides by de novo or salvage pathway. Unfused B cells die as they have a short life span. In this way, only the B cell-myeloma hybrids survive. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubated medium is then diluted into multiwell plates to such an extent that each well contains only 1 cell. Then the supernatant in each well can be checked for desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are known as monoclonal antibodies.