Development of a microtitre plate enzyme immunoassay for the determination of progesterone

A rapid, solid-phase microtitre plate enzyme immunoassay (EIA) for progesterone is described using progesterone 3-O-carboxymethyloxime-horseradish peroxidase as the label and an antiserum raised in rabbits to a progesterone 11 alpha-hemisuccinyl-bovine serum albumin immunogen. A competitive reaction was used with a reaction time of 2 h. Antibody-bound and free steroid were separated in a simple washing step of the antibody-adsorbed well surface. 2,2'-Azino-di-(3-ethylbenzthiazoline sulphonic acid) diammonium salt was used as the substrate with a reaction time of 1 h. A lower limit of sensitivity of 0.25 pg/well was obtained with the response being linear (logit/log) through 1000 pg/well. Results obtained by EIA and radioimmunoassay in several species gave excellent agreement (r = 0.98). This assay system allows accurate determination of progesterone in plasma with good specificity, precision and accuracy, and is suitable for the rapid assessment of luteal function and reproductive status in both clinical and research situations in a wide variety of species.