DEVELOPMENT, VALIDATION AND APPLICATION OF AN ULTRA-SENSITIVE 2-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 descri bed, all are of limited sensitivity and require the use oi isotopes. M any use polyclonal antibodies. Furthermore, a wide range oi follistati n preparations have been used as standards, complicating inter-laborat ory comparison. We now describe an ultra-sensitive two-site enzyme imm unoassay using a pair of mouse monoclonal antibodies raised against fo llistatin 288. The presence of sodium deoxycholate and Tween 20 in the diluent gave results for total (free and activin-dissociated) follist atin. The assay had a detection limit of <19 pg/ml and recovery of spi ked 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% re spectively. The intra- and interplate coefficients of variation were < 5%. An excess of activin-A (50 ng/ml) prior to assay did not affect fo llistatin recovery. Inhibin-A, inhibin-B, activin-A, activin-B and act ivin-AB had minimal cross-reactivity (<0.3%). However, follistatin 315 had a significant cross-reaction (9.9%). Serially diluted human sampl es gave dose-response curves parallel to the standard. Pooled human fo llicular fluid contained high concentrations of follistatin (similar t o 242 ng/ml). Follistatin was also found in maternal serum during preg nancy (first trimester similar to 0.8 ng/ml, third trimester similar t o 2.8 ng/ml), normal male serum (similar to 0.45 ng/ml), amniotic flui d (sixteen week similar to 3.63 ng/ml, term similar to 0.89 ng/ml), se minal plasma (2.4-30 ng/ml) and human granulosa cell conditioned media (similar to 0.44 ng/ml). Serial serum samples taken throughout the me nstrual cycle of ten women showed fluctuating follistatin concentratio ns (similar to 0.62 ng/ml) with no apparent relationship to the stage of the cycle. Interestingly, pooled serum from postmenopausal women ap peared to have higher follistatin levels than any of the normal women (similar to 1.4 ng/ml). The possible presence in certain samples of mi xtures of follistatin isoforms with different immunoreactivities poses major problems of interpretation in this and all other current follis tatin immunoassays. Further work is needed to identify the major-immun oreactive forms in different tissues and fluids. Nevertheless, the new assay has a number of advantages over previous assays and should prov e a useful tool for various clinical and physiological studies.