Fluorescein-specific hybridomas derived from primary mice exhibit more stringent growth requirements than do hybrids from pre-immune animals

Although the generation of antigen-specific hybridoma cell lines from animals which have been multiply immunized is now a routine procedure, the derivation of hybridomas following a single in vivo antigen injection has proven to be much more difficult to accomplish. We show that the addition of an interleukin-containing supernatant derived from rat spleen cells which have been stimulated with concanavalin A (ConA SN) to hybrids after the initial cloning step results in the consistently successful isolation of IgM anti-fluorescein specific hybridomas. However, addition of the same supernatant to fused cultures simultaneously with the addition of the HAT selection medium results in the loss of growing cells In contrast to the situation with primary hybridomas, the growth of secondary hybridomas is inhibited by the addition of ConA SN at the cloning step. Following successful cloning, there is a time-dependent variation in the sensitivity of all cell lines to ConA SN.