Enzyme-linked immunosorbent assay of total inhibin: direct determination based on inhibin α subunit-specific monoclonal antibodies

Abstract Objective: Inhibin circulates in various molecular weight forms. Alpha (α)-subunit-directed total inhibin immunoassays, which detect all forms of α subunits plus the α/β inhibin dimers, have been found valuable in the diagnosis and monitoring of ovarian cancer. Because of the dependency of the published methods on boiling sample pre-treatment with SDS and unavailability of a commercial assay, we developed an enzyme-linked immunosorbent assay (ELISA) for direct determination of total inhibin. Design and methods: Method development involved a pair of well-characterized inhibin α subunit-directed antibodies and determination of the effects of various assay parameters. Selection of the optimized protocol was guided by the outcome of comparative sample analysis using previously reported boiling sample pre-treatment reagents and protocols. Results: We report development of a simplified ELISA for total inhibin. Method evaluation data demonstrated acceptable analytical performance characteristics with detection limit of 2 ng/l (recombinant inhibin-A), dynamic range of 12.5–500 ng/l, and intra- and inter-assay imprecision of 2.3–4.6% and 3.3–5.1% at total inhibin concentrations of approximately 60–400 ng/l, respectively. The mean (±SD) recovery from spiked serum samples averaged 109 ± 14% and recovery in response to serial sample dilution was 99 ± 10%. Serum values by the direct method ( n = 40) correlated strongly with those obtained after sample pre-treatment by boiling with SDS ( r = 0.97). As expected, the total inhibin immunoreactivity in human follicular fluid fractionated by HPLC gel filtration in multiple immunoreactive peaks (8–250 kDa). In serum samples from postmenopausal women with ovarian cancer, the assay detected significantly higher total inhibin levels than in samples from normal postmenopausal controls. Conclusion: The development of a fast and simplified ELISA should facilitate wider investigations of pathophysiology and diagnostic potential of total inhibin measurement.