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Development and validation of a radioimmunoassay for follistatin in human serum
journal contributionposted on 1994-01-01, 00:00 authored by Chris Gilfillan, D M Robertson
Objective - Follistatin (FS/FSH-suppressing protein/activin-binding protein) is a single-chain glycoprotein, structurally distinct from inhibin, that has been shown to have inhibin-like activity in suppressing FSH secretion both in vivo and in vitro. The aim of these studies was to develop and validate a radioimmunoassay (RIA) for FS in human serum, and to describe the physiological variations of serum FS in humans. Patients - Serum was collected from normal men, and normal women in the follicular and luteal phases of the menstrual cycle. Clinical samples were also collected from male patients with hypogonadism, post-menopausal women and pregnant women in the first, second and third trimesters. Measurements - A RIA for FS in human serum was developed using antisera raised against purified bovine FS and using bovine FS as tracer and standard. Serial dilutions of serum were non-parallel to purified bovine FS standard in the RIA. The addition of sodium dodecyl sulphate (SDS 0.05%) to the assay buffer resolved the non-parallelism suggesting that the interference of serum in the RIA was an assay matrix effect. To characterize the serum FS immunoactivity further, serum was fractionated by gel filtration on Sephadex G-100. At neutral pH, FS immunoactivity eluted as a major peak in the molecular weight range > 200 kDa. In 0.1 M HCl, a second peak of FS immunoactivity eluted in the molecular weight range 30-60 kDa consistent with the known sizes of FS. This suggested that FS was dissociating from a larger complex. Fractions taken from the low molecular weight region diluted in parallel with bovine FS in contrast with fractions from the high molecular weight region which were non-parallel. It is concluded that the non-parallelism of human serum in the FS RIA is due to the binding of serum FS to an unknown high molecular weight factor. This interference is eliminated by the inclusion of 0.05% SDS in the assay buffer. The RIA in 0.05% SDS has been applied to the description of the normal and pathophysiological variations of FS in human serum. Results - There were no significant differences between FS levels in serum from hypogonadal men, normal women in the follicular phase of the menstrual cycle, post-menopausal women and pregnant women from the first, second and third trimesters. FS levels in serum of women in the luteal phase of the menstrual cycle were significantly lower than all other groups. FS levels in normal men were significantly higher than those in both luteal phase women and women in the first trimester of pregnancy (P < 0.05). Conclusions - These findings argue against a role for circulating follistatin in the control of gonadotrophin secretion and suggest that the gonads and/or conceptus are not the primary source of follistatin immunoactivity in serum. The lower follistatin levels in the luteal phase of the menstrual cycle remain unexplained.