Development and application of a two-site enzyme immunoassay for the determination of 'total' activin-A concentrations in serum and follicular fluid

Abstract

The performance of existing immunoassays and bioassays for activins is compromised by the presence of activin-binding proteins such as follistatin and α₂ macroglobulin (α₂M) in biological fluids. To overcome this problem we have developed a novel two-site enzyme immunoassay procedure for activin-A which incorporates an analyte denaturation and oxidation step. The optimized assay is sensitive (detection limit ∼10 pg/well), precise (mean within- and between-plate coefficients of variation 4·9 and 9·1% respectively) and accurate (activin-A recovery values of 102 ± 3 and 96 ± 5% for bovine follicular fluid (FF) and human serum respectively). In specificity tests, high concentrations of follistatin (500 ng/ml) and α₂M (100 μg/ml) did not interfere with the response signal to activin-A. In addition, no significant cross-reactivity was observed with a range of related molecules including inhibin-A, inhibin-B, activin-B (all n=76) from morphologically dominant follicles during the luteal phase showed that activin-A levels were positively correlated with inhibin-A (r=+0·54; Pr=+0·32; Pr= −0·09). Classification of these follicles according to oestrogenic status showed that activin-A, inhibin-A and total β subunit levels were highest in oestrogen-inactive follicles (PPin vivo and in vitro studies in a range of species including man. Journal of Endocrinology (1996) 148, 267–279