Radioimmunoassay of progesterone in crude serum extracts using tritiated and (125I) radioiodinated tracers

Two progesterone-tyraminyl (¹²⁵I) derivatives were prepared from progesterone-11-hemisuccinate and progesterone-3-0(carboxymethyl)oxime, respectively. The analytical performances of these tracers in the progesterone-anti progesterone-11-hemisuccinyl-BSA system were compared with those of tritiated radioligand assumed as a reference. For all of the 11 antisera tested much higher affinity and titers were observed in the case of homologous ¹²⁵I tracer, which however was found to involve drastic sensitivity losses. On the contrary, quite acceptable dose-response curves and affinity and titers close to those related to tritiated progesterone resulted with the heterologous tracer. These results, interpreted in terms of native ligand-tracer competitivity for the antibody sites, led to the choice of the C₃-tyraminyl derivative for the development of a radioimmunoassay method to be compared with the reference tritiated progesterone systems. The two procedures, including minor differences in incubation conditions and B/F separation, were validated for the direct assay of unpurified dried serum extracts. In both cases, satifactory levels of sensitivity, precision and accuracy were proved, the estimates obtained being in a good agreement with data from the literature. On the basis of the present results, some general consideration on the optimization of steroid radioimmunoassay with radioiodinated tracers are tentatively drawn.