Development of a direct radioimmunoassay for serum progesterone

Abstract

A direct radioimmunoassay for the measurement of progesterone in human serum is described. Progesterone 11α-hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mix anhydride method and then iodinated using chloramine-T. The radiochemical purity of different batches of125I-progesterone was greater than 95% and showed 70–75% binding with excess antibody. Progesterone 11α-hemisuccinate was coupled with BSA and injected to rabits. Antisera collected after three booster injections and having aK value of 1·1091/M was selected for the assay. Significant reduction in binding with antibody was seen when hormone free serum was used in the assay system. Various blocking agents were tried to reduce the serum effects and none of them were found satisfactory. From a series of optimization experiments, an assay was developed without the use of blocking agents. This assay used a much higher concentration of antibody along with lower amount of serum sample (50 μl). The optimized assay has a sensitivity of 0.5 nj/ml and a working range of 0.5 to 100 ng/ml. Serum samples were analyzed analysis showed good correlation between the results obtained from the present system and the DPC kit. (Y=0.93X+0.5,r=0.93, forn=25).