A sensitive and specific immunoradiometric assay for follistatin was developed using antifollistatin mouse monoclonal and rabbit polyclonal antibodies. The sensitivity of the assay was 0.5 micrograms/L, and cross-reactivities with recombinant human activin A and bovine inhibin were less than 0.1%. The intra- and interassay coefficients of variation were less than 10%, and the recovery rate was about 90% in human serum. The addition of activin A to the same sample resulted in a minimal influence on follistatin recovery, indicating that this assay system can measure the total level of activin-bound and unbound follistatin. Gel filtration analysis of human serum showed that the majority of immunoreactivity was eluted in a larger molecular size position than that of free follistatin, suggesting that the large part of follistatin is bound to other proteins, presumably activins, in serum. Using this assay, immunoreactive follistatin levels in various biological fluids and human sera were examined. The dose-response curves of porcine follicular and amniotic fluids were parallel to the standard curve, and porcine follicular fluid contained extremely high follistatin immunoreactivity (5.6 mg/L). The serum follistatin level in normal human volunteers was 13.3 +/- 4.7 micrograms/L (mean +/- SD; n = 60), with a tendency to increase gradually with age. On the other hand, the serum follistatin level was remarkably elevated in pregnant women (62.7 +/- 35.3 micrograms/L; n = 57), with a positive correlation with weeks of pregnancy. These data indicated that circulating immunoreactive follistatin is detectable in human serum, and the levels vary with physiological conditions such as aging and pregnancy.

We developed and validated a RIA for measuring serum activin A. The least detectable value of this assay was 0

A sensitive and specific immunoradiometric assay for follistatin was developed using antifollistatin mouse monoclonal and rabbit polyclonal antibodies. The sensitivity of the assay was 0.5 micrograms/L, and cross-reactivities with recombinant human activin A and bovine inhibin were less than 0.1%. The intra- and interassay coefficients of variation were less than 10%, and the recovery rate was about 90% in human serum. The addition of activin A to the same sample resulted in a minimal influence on follistatin recovery, indicating that this assay system can measure the total level of activin-bound and unbound follistatin. Gel filtration analysis of human serum showed that the majority of immunoreactivity was eluted in a larger molecular size position than that of free follistatin, suggesting that the large part of follistatin is bound to other proteins, presumably activins, in serum. Using this assay, immunoreactive follistatin levels in various biological fluids and human sera were examined. The dose-response curves of porcine follicular and amniotic fluids were parallel to the standard curve, and porcine follicular fluid contained extremely high follistatin immunoreactivity (5.6 mg/L). The serum follistatin level in normal human volunteers was 13.3 +/- 4.7 micrograms/L (mean +/- SD; n = 60), with a tendency to increase gradually with age. On the other hand, the serum follistatin level was remarkably elevated in pregnant women (62.7 +/- 35.3 micrograms/L; n = 57), with a positive correlation with weeks of pregnancy. These data indicated that circulating immunoreactive follistatin is detectable in human serum, and the levels vary with physiological conditions such as aging and pregnancy.

Abstract.Immunoreactive activin A (ir-activin A) release from cultured rat anterior pituitary cells was examined by measuring ir-activin A in culture medium by a specific radioimmunoassay. Ir-activin A release into the medium increased over 1-18 days, and reached a maximal level at 12-15 days. The basal levels of ir-activin A in the culture media were 0.70 ± 0.10 (mean ± SD), 1.30 ± 0.36 and 1.83 ± 0.44 ng/106 cells, when cultured for 6 days with 0, 2 and 10% fetal calf serum, respectively. LHRH induced an approximate 1.4-fold increase in ir-activin A release in contrast to a 40-60% inhibition with FSH, but LH did not affect the activin A release.In the presence of 12-o-tetradecanoylphorbol acetate (TPA), iractivin A release was enhanced, but no significant effect was induced by forskolin. Activin A was distinctly immunostained in cultured rat anterior pituitary cells. These results suggested that activin A release from the pituitary is modified by FSH and LHRH, and that the activation of protein kinase C may be involved in the action of LHRH. Kuramoto-cho, Tokushima 770, Japan more, activin modifies the secretion of inhibin and sex steroids from cultured rat granulosa cells [10,11], and enhances the meiotic maturation of rat oocytes and spermatogonial proliferation [12,13]. These observations suggest that activin plays important roles in the regulation of the pituitarygonadal axis.Anterior pituitary cells are controlled by various autocrine I paracrine factors produced locally, including activin, inhibin and follistatin [9,[14][15][16]. Activin f3A-subunit mRNA and f3A-subunit immunoreactivity have also been detected in rat anterior pituitary cells [15,17], but it still remains unknown whether and how anterior pituitary cells secrete mature activin A. We investigated the regulatory mechanism of immunoreactive (ir-) activin A secretion from anterior pituitary cells by a specific radioimmunoassay (RIA) for activin A.

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