Development of ELISA for Measurement of Progesterone Employing 17--OH-P-HRP as Enzyme Label

The present study was aimed to develop a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) to measure progesterone in human serum using a heterologous combination of immunogen and enzyme conjugate. The antiserum was raised against Progesterone-3-O-carboxymethyloxime bovine serum albumin (P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17--hydroxy-progesterone-3-O-carboxymethyloxime (17--OH-P-3-O-CMO) with Horseradish Peroxidase (HRP) to form 17--OH-P-3-CMO-HRP. A Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 100L enzyme conjugate along with 50L of standards in the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100L of 0.5M H2SO4 after 20min and the intensity of the color was measured using Tecan ELISA reader at 450nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The lowest detection limit of the assay was 0.2ng/mL. Cross-reaction with analogous steroids pregnenolone and 17--OH-P were found to be 6.8 and 6.1%, respectively. For other analogous steroids, it was less than 0.1%. The intra- and inter-assay coefficient of variation ranges from 4.52-7.39% and 4.65-9.55%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.91 (n=40).