Rapid isolation of monoclonal hybridoma cultures by a 'fusion-cloning' method: the requirement for aminopterin.

Hybridomas were generated by fusing the Balb/c SP2/0 myeloma-like cell line with either: (i) splenocytes from Balb/c mice immunized with foot-and-mouth disease virus (FMDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), African swine fever virus (ASFV) or pig thymocytes; or (ii) lymph node cells from cattle immunized with FMDV. If the fusion mixtures were plated in cloning medium of methyl cellulose and HAT medium, small hybridoma colonies developed which rarely survived. Fusion mixtures were then plated in liquid HT medium on to 3T3/A31 feeder layers in 75 cm2 flasks, incubated at 37 degrees C for 24 h before adding aminopterin, and incubated for a further 2 to 4 days before cloning in methyl cellulose/HT medium. Without the aminopterin in the cloning medium, colonies of hybridomas, which could be cultured, developed from the majority of fusions. These colonies were isolated in HT medium over feeder layers and given two subcultures in HAT medium as a precaution against any reversion to aminopterin sensitivity during the cloning. No evidence of such reversions were seen, and recloning results suggested that the initial cloning was highly efficient at generating monoclonal cultures.