Expression of defined idiotypes throughout the BALB/c anti-fluorescyl antibody response: Affinity and idiotype analyses of heterogeneous antibodies

Heterogeneous BALB/c anti-fluorescyl antibodies were shown to display increases (> 50-fold) in binding affinity from the primary through the tertiary responses. The structural basis of such affinity maturation and the diversity exhibited by anti-fluorescyl antibodies was examined by idiotypic analysis using a panel of anti-idiotype reagents specific for seven different monoclonal antifluorescyl antibodies. Because these clones exhibited binding affinities characteristic of a secondary or hyperimmune response, it was possible to examine the mechanism of affinity maturation by determining the prevalence of the seven idiotypes (Id-4-4-20, Id-20-19-1, Id-20-20-3, Id-6-10-6, Id-20-4-4, Id-4-6-10 and Id-6-19-1) in specifically purified heterogeneous preparations with low (i.e. primary response) or high (i.e. secondary and tertiary responses) binding affinities. Four of the idiotypes were not detected in heterogeneous preparations and thus each represented less than 0.1 % of the total anti-fluorescein repertoire. Although results indicated that each of three other clones expressed unique or private idiotypic determinants not present in the heterogeneous population, these idiotypes (Id-4-4-20, Id-6-10-6, Id-6-19-1) were detected and ranged from ∼0.2 to 2.0% of the repertoire. However, results indicated that each clone expressed unique or private idiotypic determinants not present in the heterogeneous population. Determinants expressed by such high-affinity monoclonal antibodies were expressed equally in all heterogeneous preparations examined. Because those determinants which were expressed were found in either low- or high-affinity heterogeneous antibodies, it is likely that the higher affinities exhibited by monoclonal antibodies derived from a secondary response are associated with unique idiotypic determinants which were not detected in polyclonal preparations. Hence, the process of affinity maturation may find as its structural correlate a mechanism such as somatic mutation which generates individual or unique idiotypes.