Development, validation and application of a two-site enzyme-linked immunosorbent assay for activin-AB

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Abstract
Monoclonal antibodies, specific for the βa and βb subunits of activin, were used to develop a new two-site ELISA for activin-AB. The assay had a detection limit of 0·19 ng/ml. High concentrations of activin-AB were found in bovine, ovine and porcine follicular fluids (FF), with less in human FF (1310, 1730, 688 and 7 ng/ml respectively). Recovery of spiked activin-AB standard from human, bovine and ovine FFs and from homogenized human placental extracts averaged 91%, 115%, 115% and 94% respectively. Within-plate coefficients of variation for different concentrations of activin-AB were between 1·3% and 2·67%. The between-plate coefficient of variation was 5·5%. Crossreactivity experiments showed the high specificity of the assay for activin-AB, with inhibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reacting 700 000 indicating its association with higher molecular weight binding protein(s). By contrast, after denaturation, immunoreactive activin-AB was detected with an Mr of ∼25 000 consistent with the complete dissociation from binding proteins. Activin-A was detected in relatively high concentrations in human FF (∼5 ng/ml), homogenized placental extracts (4·35–95·5 ng/g), sera from pregnant women (>4 ng/ml) and amniotic fluid (3–13 ng/ml), and in much lower concentrations in postmenopausal serum (500 pg/ml), normal cycle serum (100–200 pg/ml), serum from gonadotrophin-treated women (200 pg/ml) and normal adult male serum (225 pg/ml). Activin-A was also found in the culture media from explants of human amnion, chorion, maternal decidua and placenta. In marked contrast, activin-AB was undetectable (Journal of Endocrinology (1997) 153, 221–230

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