A Two-Site Enzyme-Linked Immunosorbent Assay for Inhibin1

Abstract

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 micrograms/ml (ELISA) compared with 41.0 micrograms/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.

Activins and Activin Receptors in Cell Growth

“…Recently, several two-site assays have brought the solution to this problem. Both the two-site immunoradiometric assay (IRMA) and two-site enzymelinked immunosorbent assay (ELISA) use two antibodies, one raised against the N-terminal region of inhibin a subunit, and the other specific for the C-terminal region of the inhibin PA subunit (26,27). The specificity of the assays was highly increased with a cross-reaction with free 01 inhibin subunit of less than 0.1%.…”

Effect of immunization against synthetic peptide sequences of bovine inhibin -subunit on ovulation rate and twin-calving rate in heifers

“…The ovary secretes large amounts of 'free' inhibin asubunit (Knight etal, 1989;Sugino«fa/., 1989) and other precursor forms of inhibin containing the a-inhibin sequence (Robertson et al, 1989;Sugino et al, 1989). Dimeric and biologically active inhibin may represent only a very small fraction of the total immunoreactive inhibin present in follicular fluid (Beard et al, 1991;Betteridge and Craven, 1991). Groóme et al (1990) found that a polyclonal antibody raised in sheep against the a-aminoterminal end [a-(l-29)YG] of bovine inhibin demonstrated a 29-fold greater reactivity (on a molar basis) with the immuniz¬ ing peptide than with the native 32 kDa form.…”

Cyclic Expression of HLA Class I and II Molecules on the Surface of Purified Human Spermatozoa and Their Control by Serum Inhibin B Levels

“…Several assays have been developed to measure circulating inhibin levels, such as RIA [15] or ELISA [16], but they were either not sensitive enough or unable to discriminate between inhibins A and B levels. It was not until very recently that Groome and colleagues developed a two-site assay utilizing monoclonal antibodies to both the ␣ and ␤ subunits, to specifically measure inhibin A and inhibin B in a sensitive and reliable manner [17].…”