A 2-SITE ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR INHIBIN

The investigation of the role of inhibin in the regulation of fertility is hindered by the lack of a routine, specific assay. The pituitary cell bioassay is time-consuming and the existing RIAs, based on either purified bovine 32-kDa inhibin or synthetic alpha-subunit peptides, are not specific for the biologically active inhibin molecules. We have used monoclonal antibodies, one specific for the N-terminal region of the human inhibin alpha-chain, and the other raised to a peptide sequence close to the C-terminal of the human beta-A-inhibin chain, to create a two-site sandwich ELISA specific for alpha-beta-inhibin molecules. This was used to estimate levels of inhibin in crude bovine and human follicular fluids and fractions concentrated from them. Comparison of the values obtained with the ELISA and those obtained with the pituitary cell bioassay, suggests that the ELISA measures biologically active inhibin. Compared with the peptide-based RIA, the ELISA gave much lower (as little as 100-fold lower) values for the inhibin content of these samples, e.g., bovine follicular fluid 0.375-mu-g/ml (ELISA) compared with 41.0-mu-g/ml (RIA). Such large differences, possibly due to the presence of relatively large amounts of biologically inactive forms of inhibin such as the pro-alpha-c or free alpha-forms, suggest that those RIAs, which essentially measure the level of alpha-inhibin, considerably overestimate the levels of the active forms of inhibin in the samples and that results obtained using these assays may need reinterpretation.