Methodological considerations for penicillin radioimmunoassay.

The objective of this study was to identify and define factors that compromise the utility of radioimmunoassay for quantitation of beta-lactam antibiotics in biological aqueous fluids. Serum containing antibodies to benzylpenicillin coupled as a hapten to keyhole limpet hemocyanin was used as the assay primary antibody. Two significant factors that limited the utility of the assay were the tendency for penicillin to hydrolyze in aqueous solutions, resulting in a mixture of immunorecognizable forms; and in the hydrolyzed state, to bind covalently with matrix proteins. Assay sensitivity, crossreactivity, and assay binding parameters varied with the state of hydrolysis of penicillin in tracer, standards, and unknowns and with the composition of the assay buffer. Hydrolysis of the beta-lactam ring of penicillin immediately before assay by addition of 0.1 N NaOH or penicillinase resulted in improvements in assay repeatability and uniformity by forming predominantly the penicilloate form of the compound, which was immunologically well recognized by the antibody. Nonspecific binding of penicillin (and derivatives) to proteins in biological fluids such as milk or assay buffers was shown to be a possible cause of error in the immunoassay of penicillins.