The role of metabolism in the immunogenicity of drugs: production of antibodies to a horseradish peroxidase generated conjugate paracetamol.

The allergic response to small chemically inert molecules in thought to require their enzymatic conversion to reactive metabolites which are then endowed with the capacity to bind covalently to host proteins and produce immunogenic hapten-carrier conjugates. In contrast to previous studies in which hapten-carrier conjugates have been generated following chemical modification of drugs we have examined the immunogenicity of paracetamol following direct conjugation to carrier proteins with horseradish peroxidase (HRP). Highly substituted conjugates of paracetamol with keyhole limpet haemocyanin (KLH) or bovine serum albumin (BSA) were generated using HRP. The KLH conjugate was used to immunize Balb/C mice. IgM and IgG (predominantly IgG1) responses were observed and shown by enzyme-linked immunosorbent assay (ELISA) to be hapten-specific. Manipulations of HRP levels permitted substitution of KLH to varying extents with paracetamol. Such conjugates were tested for their ability to induce a hapten-specific immune response. It was determined that substitution of 1 mol of KLH with 700 mol of paracetamol was sufficient to generate an anti-hapten response. These data suggest a mechanism by which protein-non-reactive drugs may be rendered immunogenic and provide a method for demonstrating the presence of serum antibodies reactive with drug metabolites.