Induction of covalent binding antibodies

Covalent interactions between antibody combining site residues and substrates have been implicated in the catalytic activity of abzymes elicited by design or occurring naturally in autoimmune disease. In this study, the potential for covalent binding by antibodies (Abs) was investigated by the induction of immune responses against molecules presenting chemically reactive haptenic groups. Immunogenic conjugates containing a phosphonate diester or a pyruvate carbonyl group were used to elicit antibodies that could specifically react with the electrophilic moieties. Products formed by covalent binding were detected by a western blot technique or by differential ELISA on reduced or unreduced carbonyl haptens. Antisera to the diphenylphosphonate contained antibodies with covalent reactivity, which increased with immunization. The reactivity was specific to the anti-phosphonate response and not to control immune sera induced against the unmodified carrier protein. Reactivity was focused on the antibody light (L) chain. Antisera to the phenylpyruvate hapten appeared to bind strongly to proteins modified by the carbonyl group hapten. However, anti-carrier antisera and non-immune sera had similar reactivity, indicating that the pyruvate moiety reacts nonspecifically with immunoglobulins. This suggested that affinity maturation of antibodies for reversible binding through hemiacetal or Schiff base adducts with antigens requires a less reactive carbonyl in the antigen. On the other hand, the induction of antibodies with enhanced nucleophilic reactivity toward phosphonate esters implies that irreversible binding to the B cell receptor can drive clonal expansion and antibody selection. These results support a designer strategy for generating nucleophilic abzymes and could also account for the occurrence of chemically reactive or catalytic antibodies in natural immunity or autoimmunity.