Isolation and Characterization of Distinct Antibody Populations from Antisera Directed against Idiotypes of Rabbit Homogeneous Antibodies

Abstract

A hapten, 3-0-α-N-acetylgalactosaminosyl-N-acetylgalactosamine, was found to inhibit the binding of eight idiotypic antisera directed against different homogeneous antistreptococcal Group C polysaccharide antibodies. The sensitivity of the different idiotypic reactions to inhibition by a given concentration of hapten was found to be quite variable, ranging from 20 to 80% inhibition. This result suggested that idiotypic antisera contained two populations of antibodies: those which are reactive with determinants in the antigen-binding site and those directed against nonbindingsite idiotypic determinants. This suggestion was verified by fractionation of idiotypic antisera on affinity chromatography columns consisting of homogeneous antibodies attached to Sepharose. The absorbed idiotypic antibodies were separated into two populations by successive elution with the Group C hapten and ammonium thiocyanate. The antibody fraction obtained by elution with the hapten comprised approximately 33% of the total idiotypic antibody recovered from the column. The hapten-eluted fractions from different idiotypic antisera varied in their sensitivity to hapten inhibition although each could be inhibited to a greater extent than the original antiserum or the ammonium thiocyanateeluted fraction. One of the hapten anti-idiotype fractions appeared to be homogeneous by isoelectric focusing whereas the other hapten and ammonium thiocyanate-eluted fractions contained multiple bands. Kinetic analysis revealed that idiotypic reactions of antibody fractions obtained by hapten elution were competitively inhibited by the hapten, although the mechanism by which the competition occurs was not completely resolved by this analysis.