After the diethyl ether extraction, the remaining Ringer’s solution was used for ET-1 extraction. BSA was added to the samples to a final concentration of 1 mg/ml. Follicular fluids (2 ml) were diluted with the same volume of distilled water, and the pH was adjusted to 2.5 with acetic acid. The samples were then applied to a Sep-Pak C18 Cartridge (Waters, Millford, MA) as described previously. The residue was evaporated and then dissolved in 200 ^l assay buffer (42 mM Na2HPO4, 8 mM KH2PO4, 20 mM NaCl. 4.8 mM EDTA, 0.05% BSA, pH 7.5) for peptide EIA. Thus, the samples were concentrated about 15-fold as a result of the process. The recovery rate of ET-1 (10 pg/ml) that had been added to the Ringer’s solution was 63%.
The concentrations of the various hormones were determined in duplicate by double-antibody EIAs using 96-well ELISA plates (Corning Glass Works, Corning, NY). buy antibiotics online
The EIA for P4 was done as previously described. The standard curve ranged from 0.05 to 25 ng/ml, and the ED50 of the assay was 2.8 ng/ml. The average intra- and interassay coefficients of variation (CVs) were 5.8% and 8.2%, respectively.