A SOLID-PHASE ENZYME-IMMUNOASSAY FOR THE DETERMINATION OF PROGESTERONE IN BOVINE, PORCINE AND OVINE PLASMA

The purpose of this project was to develop a valid quantitative enzyme immunoassay (EIA) for progesterone in blood plasma of cattle, pigs and sheep. Rabbit anti-progesterone, mouse monoclonal anti-rabbit IgG, au thentic progesterone, and acetylcholine esterase bound covalently to p rogesterone were the principal reagents used to develop the EIA. Ninet y-six well microtiter plates were coated with mouse monoclonal anti-ra bbit IgG and saturated with bovine serum albumin before use. Rabbit an ti-progesterone was diluted to a working dilution of 1:2.0 x 10(6). St andard curves were linear and ranged from 1.56 to 400 pg of progestero ne per well which allowed for the measurement of 0.03125 to 8.0 ng mL( -1). Assay sensitivity averaged 1.56 pg well(-1). Progesterone was ext racted from plasma samples with petroleum ether. Plasma samples (n = 3 or 4 from each species) with unknown amounts of progesterone that wer e extracted and serially diluted with EIA buffer did not deviate from parallelism with progesterone standard curves in buffer. The correlati on between EIA and radioimmunoassay (RIA) measurements of progesterone in the same plasma samples was high (P < 0.0001) for all three specie s (r = 0.96 for bovine; r = 0.96 for porcine; r = 0.94 for ovine). The regression of EIA data on RIA data produced the following equations: Y = 0.70 + 0.84x, with r(2) = 0.92 for bovine; Y = -0.72 + 0.93x, with r(2) = 0.93 for porcine; and Y = -0.21 + .96x, with r(2) = 0.89 for o vine. The intra- and inter-assay coefficients of variation were 5.4 an d 10.6% for bovine, 5.8 and 11.0% for porcine and, 6.1 and 12.3% for o vine, respectively. These data show that this EIA is a valid and relia ble method for quantitating progesterone in extracts of bovine, porcin e and ovine plasma.