Biological and immunological characterization of inhibin forms in human plasma.

Abstract

A number of immunoassay methods have been developed recently to detect specifically the bioactive alpha-beta A subunit inhibin dimer (inhibin A) in human plasma. However, the specificity of these assays in terms of their ability to detect the range of inhibin forms found in plasma and their relationship to bioactivity have not been investigated. Inhibin was fractionated from human follicular fluid (hFF) and serum/plasma from women stimulated with gonadotropins (IVF serum), and from postmenopausal and male plasma, using a combined immunoaffinity/preparative SDS-PAGE procedure. The molecular weight profile of inhibin was established by inhibin in vitro bioassay, three alpha-beta A subunit specific immunoassays, and three alpha subunit-directed immunoassays that detect the alpha subunit as well as inhibin A and B forms. In hFF inhibin forms of 33, 36, 55 and 66K were detected by in vitro bioassay and by most immunoassays except for 33 k inhibin, which was nondetectable by one alpha-beta A ELISA. The alpha subunit-directed assays also detected activity in the 29-31K region, in some assays in considerably high levels. In IVF serum in vitro bioactivity and immunoactivities were detected between 27 and 100K with the alpha-beta A assays failing to detect all bioactive forms. Alpha subunit-directed assays gave similar immunoactive profiles. Neither in vitro bioassay nor alpha-beta A assays detected activity in post-menopausal plasma or male plasma, while alpha subunit-directed assays showed peaks predominantly at 36 k, although at low levels. It is concluded that dimeric inhibin A specific assays detected bioactive inhibin forms in hFF and to a lesser extent in IVF serum. Alpha subunit-directed assays correlated poorly with in vitro bioassay in hFF because of the high alpha subunit levels in this sample. The higher correlation between these assays in IVF serum suggested that there was little free alpha subunit. The 36K form in male plasma may be free alpha subunit or inhibin B.

Intra-pituitary regulation of gonadotrophs in male rodents and primates

“…Immunoreactive inhibin was subsequently reported to decline coincident with the rise in FSH (Ackland & Schwartz 1991). Although the inhibin immunoassay used did not discriminate between the bioactive inhibin a-b dimer and the uncombined inhibin-a subunit that circulates in excess, results with specific two-site ELISAs for inhibin-B (Woodruff et al 1996, Sharpe et al 1999 tend to confirm those findings.…”

“…Inhibin is composed of an a-subunit disulfide linked to one of two b-subunits, the b A -subunit to form inhibin-A, or the b B -subunit to form inhibin-B (Ying 1988); inhibin-B is also the form secreted by the testis (Anawalt et al 1996, Woodruff et al 1996. Inhibin a-subunit mRNA is found in the rat pituitary in low concentration using solution hybridization methods (Bilezikjian et al 1996) but was undetectable by northern hybridization in monkey pituitary RNA (Attardi et al 1992).…”

“…Interestingly, however, circulating inhibin levels in rats and monkeys tend to overlap (Sharpe et al 1999). Perhaps because the current two-site inhibin-B assays utilize antisera against the human inhibin subunits, and there are large-molecular-weight forms of circulating inhibin-B of uncertain bioactivity, at least in primates (Robertson et al 1996, Winters & Plant 1999, immunoassay detection and specificity may differ in primates and rats. If so, the greater rise in FSH following castration or immunoneutralization of inhibin in male primates could reflect higher circulating levels of bioactive inhibin-B.…”

L'Inhibine B chez les blessés radiculo-médullaires. Résultats préliminaires

“…L'isolement et la caract6risation des inhibines ont 6t4 laborieux [22], quelques m 6 t h o d e s de dosage peu sp6cifiques ont e n t r e n u des doutes sur la physiopathologie de cette h o r m o n e [7]. I1 a fallu a t t e n d r e 1993 pour disposer d' u n dosage sp6cifique des diff4rentes formes de l'inhibine (inhibine B, inhibine A, sous-unit6 de l'inhibine) [18,6,19]. Ces dosages p a r m 6 t h o d e ELISA, outre l e u r bonne sp6cificit6 se sont m o n t r 6 s tr6s fiables et ont p e r m i s de nomb r e u s e s 6tudes soit chez l'homme, soit chez la f e m m e [20].…”

Comparison of Inhibin A Immunoassays: Recommendation for Adoption of Standardized Reporting

“…The first to be developed is manufactured by Serotec, Inc. This method is not been cleared by the US Food and Drug Administration for clinical use but has been used in an investigational context to measure inhibin A in various clinical conditions (15,16 ). Because of the difficulties in assay development, the Serotec inhibin A ELISA represents the method on which much of our current knowledge concerning dimeric inhibin A concentrations in human blood is based.…”