Hybridomas producing hemolytic plaques used to study the relationship between monoclonal antibody affinity and the efficiency of plaque inhibition with increasing concentrations of antigen

Abstract

Hybridoma clones producing anti-hapten antibodies were established by fusing spleen cells from mice immunized with 4-azophthalate keyhole limpet hemocyanin to drug-resistant non-producing myelomas. The affinity and homogeneity of the immunochemically purified anti-phthalate antibodies were determined by equilibrium dialysis. We report here a broad distribution of the monoclonal antibody affinities ranging from a low of 4.0 X 10(4) to a high of 4.0 X 10(7) (L/M). The binding data for eleven anti-phthalate antibodies described a straight line in a Scatchard plot as would be expected for homogeneous antibodies. We determined that all hybridomas, except those secreting very low affinity antibody, produced hemolytic plaques. The hybridomas made it possible to test several assumptions regarding the association of plaque morphology and plaque inhibition with the affinity of the antibody secreted by the plaque-forming cells. Our studies indicate that the affinity of the antibody secreted by the hybridoma clones does not correlate with either the size of the plaque or with the efficiency with which the hybridoma-produced plaques are inhibited by free hapten. By comparing hybridoma-produced plaques to plaques produced by phthalate-immune spleen cells it has been demonstrated that the plaque size within each hybridoma clone was substantially less heterogeneous than that observed for the immune spleen cells. Our results do support the assumption that the range of free hapten concentration over which PFC are inhibited is a reflection of PFC heterogeneity. The analysis of the PFC inhibition of hybridomas produced an interesting and unexpected result which is reported here. It was observed that subinhibitory doses of free hapten caused an enhancement in the number of hybridoma-produced plaques. The relevance of this finding to the recent observation and interpretation of plaque enhancement (i.e., the displacement of anti-idiotype antibodies) observed for immune spleen cells is discussed.