Behavior of the idiotypic network in conventional immune responses. III. Detection and enumeration of cells producing idiotypic and anti-idiotypic antibodies by a spot ELISA technique.

The idiotypic (Ab1) and anti-idiotypic (Ab2) responses of spleen cells of mice immunized with DNP-Ficoll were assayed with the spot ELISA technique in wells coated with DNP-BSA or with affinity-purified rabbit anti-DNP antibodies, respectively. In agreement with results previously obtained with the hemolytic plaque technique, large numbers of Ab1 and Ab2 ELISA spots were found. The cytokinetics of the Ab1 and Ab2 responses were very similar and the peak responses occurred simultaneously. The isotypes of Abs1 and Abs2 were also similar. Both responses were specifically inhibited by soluble DNP-lysine. Similar results were obtained with spleen cells from mice immunized with the T-dependent antigen DNP-KLH and with the T-independent antigen fluorescein-Ficoll. In contrast, no Ab2 response was detected when spleen cells from mice immunized with ovalbumin were assayed, probably because the affinity-purified rabbit anti-ovalbumin antibodies used to coat the wells for the Ab2 assay contained molecules of several specificities, corresponding to the several epitopes of the ovalbumin molecule. The many similarities between the Ab1 and Ab2 responses to the haptens suggest that they reflected two different reactivities of the same antibody molecules.