Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

Recent studies have found follistatin to be an important regulator of activin bioactivity. Whilst a number of assay formats have been described, all are of limited sensitivity and require the use of isotopes. Many use polyclonal antibodies. Furthermore, a wide range of follistatin preparations have been used as standards, complicating inter-laboratory comparison. We now describe an ultra-sensitive two-site enzyme immunoassay using a pair of mouse monoclonal antibodies raised against follistatin 288. The presence of sodium deoxycholate and Tween 20 in the diluent gave results for total (free and activin-dissociated) follistatin. The assay had a detection limit of <19 pg/ml and recovery of spiked follistatin 288 from amniotic fluid, serum seminal plasma, human follicular fluid and granulosa cell conditioned medium averaged 100.7 +/- 7.5%, 89.1 +/- 5.5%, 98 +/- 4.9%, 96 +/- 7.2% and 123.9 +/- 11% respectively. The intra- and interplate coefficients of variation were < 5%. An excess of activin-A (50 ng/ml) prior to assay did not affect follistatin recovery. Inhibin-A, inhibin-B, activin-A, activin-B and activin-AB had minimal cross-reactivity (<0.3%). However, follistatin 315 had a significant cross-reaction (9.9%). Serially diluted human samples gave dose-response curves parallel to the standard. Pooled human follicular fluid contained high concentrations of follistatin (approximately 242 ng/ml). Follistatin was also found in maternal serum during pregnancy (first trimester approximately 0.8 ng/ml, third trimester approximately 2.8 ng/ml), normal male serum (approximately 0.45 ng/ml), amniotic fluid (sixteen week approximately 3.63 ng/ml, term approximately 0.89 ng/ml), seminal plasma (2.4-30 ng/ml) and human granulosa cell conditioned media (approximately 0.44 ng/ml). Serial serum samples taken throughout the menstrual cycle of ten women showed fluctuating follistatin concentrations (approximately 0.62 ng/ml) with no apparent relationship to the stage of the cycle. Interestingly, pooled serum from postmenopausal women appeared to have higher follistatin levels than any of the normal women (approximately 1.4 ng/ml). The possible presence in certain samples of mixtures of follistatin isoforms with different immunoreactivities poses major problems of interpretation in this and all other current follistatin immunoassays. Further work is needed to identify the major immunoreactive forms in different tissues and fluids. Nevertheless, the new assay has a number of advantages over previous assays and should prove a useful tool for various clinical and physiological studies.