Studies on the Mechanism of the Formation of the Penicillin Antigen

An excess of D-benzylpenicillenic acid (BPE) was reacted with human gamma-globulin, human serum albumin, gelatin, and poly-L-lysine in aqueous solution buffered at pH 7.5-8.0. Under these conditions, BPE reacted predominantly with lysine epsilon-amino groups of the proteins to form the mixture of diastereomers of epsilon-N-(D-alpha-benzylpenicilloyl)-lysine groups (Di-BPO-Lys). BPE reacted also, but to a considerably smaller extent, with cystine disulfide linkages of human gamma-globulin and human serum albumin to form D-benzylpenicillenic acid-cysteine mixed disulfide groups (BPE-SS-Cys). Conjugates containing large numbers of BPE or D-penicillamine mixed disulfide groups were prepared by reaction of BPE or D-penicillamine with thiolated human gamma-globulin under mild oxidizing conditions. Anti-penicillin antibodies were produced in rabbits by immunization with either potassium penicillin G (PG) or a preincubated mixture of PG with normal rabbit serum (PG-NRS) in complete Freund's adjuvant. Specific precipitation analyses in aqueous and gel media (Ouchterlony), PCA analyses, and specific inhibition of these reactions with haptens were carried out on the rabbit anti-PG and anti-(PG-NRS) sera, using the above conjugates as antigens. The anti-penicillin antibodies were found to be directed against the diastereomeric mixture of N-(D-alpha-benzylpenicilloyl) groups, predominantly the Di-BPO-Lys groups. By these techniques, no antibodies directed against the BPE-mixed disulfide or the D-penicillamine mixed disulfide groups were detected. Three out of six patients with histories of allergic reactions to PG responded with wheal-and-erythema reactions to the N-(D-alpha-benzylpenicilloyl) (BPO) groups contained in BPE-human gamma globulin conjugate. Another such patient exhibited serum antibodies specific for the BPO group. One patient being treated with 25 gm per day of PG showed the presence of non-dialyzable antigenic BPO-conjugates in his serum. These results demonstrate that the diastereomeric BPO groups (predominantly Di-BPO-Lys groups) are major antigenic determinant groups responsible for PG hypersensitivity in rabbits and human beings. The possible clinical usefulness of multivalent Di-BPO conjugates and univalent Di-BPO haptens is discussed.