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#hybridoma selection

2 public questions tagged with this topic.

Immunological Applications

Aminopterin in HAT medium functions by:

Aminopterin classifies as antifolate antimetabolite structurally analogous to folic acid with pteridine ring substitution preventing enzymatic reduction. Dihydrofolate reductase normally catalyzes NADPH dependent reduction of dihydrofolate to tetrahydrofolate, central one-carbon carrier crucial for biosynthesis. Tetrahydrofolate derivatives required at two distinct steps: 10-formyl tetrahydrofolate donates formyl groups to glycinamide ribonucleotide transformylase and aminoimidazole carboxamide ribonucleotide transformylase in de novo purine pathway synthesizing inosine monophosphate precursor

Ref: Goodman & Gilman Pharmacol 13th ed DHFR antifolate; Lodish MBoC 9th ed Fig 7-32 aminopterin blocks de novo purine thymidylate.

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Basic Applied Biology

HAT medium selects hybridoma cells based on:

HAT medium selection developed by John Littlefield in 1964 exploits differential nucleotide anabolism pathways to isolate hybrid cells from mixture of parental populations. De novo purine and pyrimidine synthesis requires tetrahydrofolate-dependent enzymes, specifically aminopterin, a folate analog, competitively inhibits dihydrofolate reductase DHFR blocking de novo pathway. Alternative salvage pathway recycles hypoxanthine via hypoxanthine-guanine phosphoribosyltransferase HGPRT converting to IMP and thymidine via thymidine kinase TK to TMP, bypassing blocked de novo routes. Myeloma partners

Ref: Littlefield Science 1964 Selection of hybrids; Abbas Immunology 10th ed. HAT selection; Alberts Ch nucleotide metabolism salvage.

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