DEVELOPMENT, VALIDATION AND APPLICATION OF A 2-SITE ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR ACTIVIN-AB

Monoclonal antibodies, specific for the beta A and beta B 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 act ivin-AB were found in bovine, ovine and porcine follicular fluids (FF) , with less in human FF (1310, 1730, 688 and 7 ng/ml respectively). Re covery 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 diff erent concentrations of activin-AB were between 1.3% and 2.67%. The be tween-plate coefficient of variation was 5.5%. Crossreactivity experim ents showed the high specificity of the assay for activin-AB, with inh ibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reac ting <0.2%. Incubation with high concentrations of follistatin (500 ng /ml) prior to assay did not affect the recovery of activin-AB, Samples of bovine, porcine, ovine and human FF gave dose responses parallel t o that of the standard, as did bovine granulosa cell-conditioned media . In human and porcine FF, levels of activin-A and activin-AB were sim ilar whereas, in bovine and ovine FF, activin-A levels were approximat ely threefold higher than activin-AB levels. As we have reported previ ously for activin-A, nearly all of the endogenous activin-AB in bovine FF was detected in the eluate from gel permeation chromatography with an M-r of >700 000 indicating its association with higher molecular w eight binding protein(s). By contrast, after denaturation, immunoreact ive activin-AB was detected with an M-r of similar to 25 000 consisten t with the complete dissociation from binding proteins, Activin-A was detected in relatively high concentrations in human FF (similar to 5 n g/ml), homogenized placental extracts (4.35-95.5 ng/g), sera from preg nant women (>4 ng/ml) and amniotic fluid (3-13 ng/ml), and in much low er concentrations in postmenopausal serum (500 pg/ml), normal cycle se rum (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 de cidua and placenta. In marked contrast, activin-AB was undetectable (< 0.19ng/ml) in all of these samples with the exception of human FF (sim ilar to 7 ng/ml). In conclusion, we have developed a sensitive and spe cific ELISA to measure total (bound+free) activin-AB. Preliminary resu lts show a more restricted distribution of this isoform compared with activin-h. The presence of high levels ofboth activin-A and activin-AB in FF suggests a function for both isoforms in the developing ovarian follicle.