A direct antigen heterologous enzyme immunoassay for measuring progesterone in serum without using displacer

An antigen heterologous enzyme-linked immunosorbent assay (ELISA) for directly measuring progesterone in serum is described. Six combinations of antigens and enzyme conjugates were tested; the enzyme conjugate 17-alphaOH-progesterone-3-O-carboxymethyloxime-alkalinephosphatase (17-alphaOH-P-3-CMO-ALP) and the immunogen progesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) were found to be best. Fifty microliters of standard or serum sample and 100 microL of the 17-alphaOH-P-3-CMO-ALP enzyme conjugate were added to the antibody coated wells, and incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using p-nitrophenyl phosphate as substrate. The sensitivity of the assay was 0.11 ng/mL, and intra- and inter-assay CVs ranged from 5.1% to 9.6%. The analytical recoveries were 97-105%. The serum progesterone values obtained by this method correlated well with those obtained by radioimmunoassay; r=0.97 (n=44). Moreover, in this ELISA no displacing agent was used or special means was required to displace progesterone from corticosteroid binding globulin (CBG). Serum progesterone concentrations of subjects, with histories of recurrent spontaneous abortions were also measured, and correlated well with clinical history.