Development of ELISA for measurement of progesterone employing 17 - OH-P-HRP as enzyme label

Abstract

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.