Rabbit antisera prepared against conjugates of the benzylpenicilloyl (BPO) bifunctional haptenic group were analyzed to determine whether the antibodies are adapted to only a portion of the large BPO molecule, or to the entire molecule, and whether specificity extends to the lysine side chain and adjoining structures of the immunizing carrier protein.
No antibodies adapted to the phenylacetylamine portion of the BPO group could be detected in a pooled rabbit anti-BPO serum globulin fraction by PCA and quantitative precipitin analysis using several phenylacetylamine-protein conjugates as antigens. No antibodies adapted only to the thiazolidine carboxylic acid portion of the BPO molecule were detected in the anti-BPO globulin fraction using quantitative precipitin and hapten inhibition methods. At least the bulk of the anti-BPO antibodies was found to be adapted to the entire BPO haptenic group.
By quantitative hapten inhibition of precipitation of the anti-BPO globulin fraction, the anti-BPO antibodies were found to show specificity for a 6 carbon amide side chain corresponding to the lysine side chain through which BPO groups are bound predominantly to protein. The contribution of this 6 carbon chain to antibody-hapten binding was small; (–ΔF°) was calculated to be 460 calories per mole (average).
Rabbit anti-BPO antibodies prepared against BPO-rabbit serum albumin conjugates showed specificity also toward structures of the immunizing carrier protein, and possibly toward secondary or tertiary structural configurations. Penicilloyl conjugates of rabbit serum albumin precipitated from 3 individual rabbit antisera more anti-BPO antibodies than did penicilloyl conjugates of heterologous carriers (poly-L-lysine, human serum albumin, and human γ-globulin).
Anti-BPO antibodies demonstrated heterogeneity with regard to closeness of fit to the haptenic group, or with regard to the dimensions of the combining sites, or both.
It was concluded that at least a large part of anti-BPO antibodies are specifically adapted to a large antigenic unit comprised of the entire BPO group, the lysine side chain, and structural configurations of the immunizing carrier protein.