The Heterogeneity of Combining Sites of Anti-Benzylpenicilloyl Antibodies Obtained from Individual Rabbits: Fractionation of Antibodies with a Specific Immunoadsorbent

Abstract

Benzylpenicilloyl (BPO) groups were introduced into the bisdiazotized benzidine treated insoluble bovine γ-globulin (BGG) aggregates by direct coupling of benzylpenicillin in alkaline condition. The insoluble BPO-BGG aggregates were used for the analysis of site heterogeneity of rabbit anti-BPO-RSA antibody as a specific immunoadsorbent. The radioiodinated γG fraction prepared from a single rabbit antiserum was charged on the immunoadsorbent and eluted step by step with five haptens corresponding to the constituent of the BPO molecule. Five different antibody fractions were separated. By rechromatography these fractions were demonstrated to be specifically adapted to phenylacetic acid, phenylacetylglysine, 6-acetyl-aminopenicilloic acid, benzylpenicilloic acid and benzylpenicilloyl-ε-aminocaproate respectively. The order of binding affinity to the immuno-adsorbent of these five different antibody populations seemed to reflect the order of the sizes of combining region. The cross-reactivity of anti-BPO-RSA antibody with penicilloyl derivatives having different acyl side chain was examined. The fraction directed to the nuclear portion of the BPO molecule was shown to react as strongly with penicilloyl derivatives of synthetic penicillins as with BPO-EACA. In contrast, the fraction directed to the acyl side chain portion of the BPO molecule was shown to have various cross-reactivity with penicilloyl derivatives of synthetic penicillins. The cross-reactivity decreased in the order of phenoxymethylpenicilloyl-, 5-methyl-3-phenyl-4-isoxazolylpenicilloyl-, 2,6-dimethoxyphenylpenicilloyl-ε-aminocaproate.