A sensitive EIA for 17 beta-estradiol and progesterone in culture medium for oocyte in vitro maturation procedures.

Abstract

A sensitive heterologous enzyme immunoassay (EIA) was validated to determine 17 beta-estradiol (E2) and progesterone levels, without previous extraction, in culture medium from rabbit oocytes matured in vitro with and without the addition of IGF-I. Polyclonal E2 (C902), and progesterone (C914) antibodies were raised in rabbits using 6-keto-17 beta-estradiol 6-carboxymethyloxime:BSA, and 11 alpha-hydroxyprogesterone 11 alpha-hemisuccinate:BSA. Horseradish peroxidase was used as label, conjugated to 17 beta-estradiol 3-hemisuccinate, and to progesterone 3-carboxymethyloxime. Standard dose response curves covered a range between 0 and 1 ng/well (100 microliters). The low detection limits of the technique were 1.99 pg/well for E2, and 13.21 pg/well for progesterone. Intra- and interassay coefficient of variation percentage (% CV) were < 6.3 and < 7.8 for E2 and progesterone, respectively (n = 10). The recovery rate of known E2 or progesterone concentrations added to a pool of culture maturation medium averaged 96.39%, and 98.65%, respectively. Compared with RIA, EIA values were in close agreement for E2 (n = 15, R = 0.96, P < 0.001), and progesterone (n = 15, R = 0.99, P < 0.001). Medium samples were obtained after oocyte maturation in vitro for 16 h. Use of IGF-I significantly elevated steroids production in the oocyte surrounded cumulus cells. The EIA described here is highly sensitive and specific assay, and provides a rapid, simple, inexpensive, and non-radiometric alternative to RIA for determining E2 and progesterone levels in oocyte culture medium.