Influence of the conjugation site on the specificity of monoclonal antibodies to progesterone and on the performance of direct enzyme immunoassay

Abstract

Monoclonal antibodies against progesterone conjugated to carrier protein through substituent on the A-ring (C3 position) or the C-ring (C11 position) of progesterone were used in the enzyme immunoassay. Antibody specificities were determined by testing the ability of 11 represensitive steroids to displace labelled progesterone in a competitive enzyme immuno-assay (EIA) and a radioimmunoassay (RIA). Immunization with progesterone conjugated to BSA through substituent on the A-ring (C3) resulted in the formation of monoclonal antibody (mAb) which fairly specific for progesterone. While immunization with progesterone at the 11-position (C11) resulted in the mAbs which were very specific for progesterone. The best EIA system was developed by using mAb against progesterone-11α-hemisuccinyl BSA and tracer of progesterone-3(O-carboxymethyl oxime)-horseradish peroxidase. Our approach is to confirm the overall orientation of the steroid in the binding site. Immunobiochemical analysis of mAb suggested that the D-ring is substantially more buried in the binding pocket than the A-ring. Our assay was designed to use the reagent inactivating cholesterol binding globulin in serum so that the extraction of the hormone into organic solvent was unnecessary. Therefore, our assay system can detect directly and rapidly the progesterone level in serum within one hour.