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Hybridoma selection using HAT medium
Hybridoma selection after fusion of myelomas and spleen cells is a critical step in monoclonal antibody production. Often scientists use the HAT (hypoxanthine-aminopterin-thymidine) method to accomplish this task.
During the fusion process, three types of cells are present: (1) unfused myeloma cells that are deficient in an enzyme called HGPRT, (2) unfused spleen cells, and (3) fused hybridoma cells.
- Unfused spleen cells are easily selected against since they do not replicate in culture.\
- Unfused myelomas can be selected against using media containing HAT. The aminopterin found in the medium blocks the de novo DNA nucleotide synthesis pathway. Typically when the de novo pathway is blocked, cells will then utilize the salvage pathway as an alternative means to replicate (only if hypoxanthine and thymidine are present). However, these myelomas are unable to do so since they are deficient in an enzyme called HGPRT, which is required for the salvage pathway. Hence, myelomas are unable to replicate in culture. \
- Only hybridomas survive. Hybridomas inherit a functioning HGPRT enzyme from the spleen cells, so even though the de novo pathway is blocked, they can still use the salvage pathway to replicate.
Please watch our tutorial video for more details.