A SIMPLE TECHNIQUE FOR THE RAPID ENRICHMENT OF CLASS AND SUBCLASS HYBRIDOMA SWITCH VARIANTS - A 1000-FOLD ENRICHMENT IN HALF THE TIME, FOR HALF THE COST

Switching parental hybrids in vitro to downstream switch variant clone s producing more desirable monoclonal antibodies (MoAbs) requires eith er labor intensive and time consuming subcloning techniques, or fluore scence activated sorting of the desired clones. We tested the hypothes is that enrichment of downstream switch variant clones might be achiev ed by selective lysis of upstream hybridoma cells followed by expansio n of the enriched downstream clone. Using a parental hybridoma with su rface and secretory IgM, we attempted to enrich downstream switch vari ant clones producing class (IgG) and subclass (IgG1 or IgG2a) MoAbs. E nrichment of downstream IgG, IgG1 and IgG2a MoAb-producing switch vari ants was achieved by single or repeated antibody-dependent, complement -mediated lysis of the upstream IgM-bearing parental hybridoma cells f ollowed by limited subcloning. Two exposures of parental hybridoma cel ls to lysis followed by plating at 100 cells/well enriched the frequen cy of switch variants up to 1235-fold, enabling the development of IgG 1 or IgG2a-producing subclones exhibiting high yield antibody producti on. Using this protocol, production time and costs were reduced by > 5 0% when compared to the standard technique. This novel technique for t he rapid isolation and expansion of switch variant clones should be id eal for most laboratories, particularly those without access to cell s orting capabilities.