Studies on the Influence of the Structural Modifications in the Tracer on the Immunoassay of Progesterone

Abstract

The main objective of the present study was to examine the influence of different bridges in radioiodinated tracers on the assay performance of progesterone using antibodies. Three homologous and two heterologous immunoassay systems for the measurement of progesterone in human serum are described. Using an antiserum raised against progesterone‐11α‐hemisuccinate–bovine serum albumin (BSA), assays with homologous radioligands, namely progesterone‐11α‐hemisuccinate‐125I‐tyrosine methyl ester (TME) and progesterone‐11α‐hemisuccinate‐125I‐histamine, heterologous bridge radioligand, namely progesterone‐11α‐hemiphthalate‐125I‐TME, and a heterologous site radioligand namely progesterone‐3‐(O‐carboxymethyl) oxime (CMO)‐125I‐histamine were optimized. A homologous assay system, using antiserum raised against progesterone‐3‐carboxymethyl oxime‐BSA and progesterone‐3‐CMO‐125I‐histamine as the radioligand was also optimized to develop a radioimmunoassay (RIA) for serum progesterone. Amongst the two homologous radioligands, viz., progesterone‐11α‐hemisuccinate‐125I‐histamine and the corresponding TME conjugate tracer, the former yielded a standard curve with a higher slope (−0.6) as compared to the latter (−0.5). The heterologous bridge system with progesterone‐11α‐hemiphthalate‐125I‐TME resulted in a more sensitive assay (slope of −0.8) than the homologous tracers, whilst the heterologous site radioligand, viz., progesterone‐3‐CMO‐125I‐histamine gave the most sensitive assay (slope of −1.2). The homologous assay with antiserum against progesterone‐3‐CMO‐BSA and progesterone‐3‐CMO‐125I‐histamine tracer gave a standard curve having a slope of –0.97. The two antibodies developed against progesterone, viz., progesterone‐11α‐hemisuccinate‐BSA and progesterone‐3‐CMO‐BSA were characterized for their titre, sensitivity, and specificity. Considering the slope, sensitivity, cross‐reactivity, and the quality of tracer, the assay system using antiserum against progesterone‐11α‐hemisuccinate‐BSA and progesterone‐3‐CMO‐125I‐histamine was found to be suitable for the development of RIA for serum progesterone. The bridges used in an immunogen for production of antibodies, as well as in the preparation of tracer, have a great influence on the assay characteristics.