Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin L W Evans, S Muttukrishna 1 and N P Groome School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, UK and 1 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK (Requests for offprints should be addressed to L W Evans, School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Headington, Oxford OX3 0BP, UK) Abstract 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%). How- ever, 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 (242 ng/ml). Follistatin was also found in maternal serum during pregnancy (first trimester 0·8 ng/ml, third trimester 2·8 ng/ml), normal male serum (0·45 ng/ml), amniotic fluid (sixteen week 3·63 ng/ml, term 0·89 ng/ml), seminal plasma (2·4–30 ng/ml) and human granulosa cell conditioned media (0·44 ng/ml). Serial serum samples taken throughout the menstrual cycle of ten women showed fluctuating follistatin concentrations (0·62 ng/ml) with no apparent relationship to the stage of the cycle. Interestingly, pooled serum from postmeno- pausal women appeared to have higher follistatin levels than any of the normal women (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. Journal of Endocrinology (1998) 156, 275–282 Introduction Follistatin is a monomeric glycosylated polypeptide chain which was initially identified in and isolated from both bovine and porcine follicular fluids on the basis of its inhibition of pituitary follicle-stimulating hormone (FSH) secretion (Robertson et al. 1987, Ueno et al. 1987, Ying et al. 1987). It is well documented that follistatin exerts its inhibitory effect on FSH secretion by neutralizing activin bioactivity (Nakamura et al. 1990, Kogawa et al. 1991, Shimonaka et al. 1991, de Winter et al. 1996). There are two main forms of mature mammalian follistatin (FS) which occur as a result of alternative modes of precursor mRNA splicing, giving core proteins of 315 amino acids and the carboxy-truncated variant of 288 amino acids (FS315 and FS288) (Shimasaki et al. 1988, Michel et al. 1990, Inouye et al. 1991). Further variations in the molecular weight of mature follistatin occur as a result of varying degrees of glycosylation (Inouye et al. 1991, Sugino et al. 1993). Previous immunoassays for follistatin have either been radioimmunoassay (RIA) or immunoradiometric assay (IRMA) formats (Table 1). The most sensitive of these assays has a detection limit of 500 pg/ml follistatin. This is two orders of magnitude less sensitive than the inhibin enzyme-linked immunosorbent assays (ELISAs) from our laboratory (Groome et al. 1994, 1995, 1996). These pre- vious follistatin assays have other disadvantages, including 275 Journal of Endocrinology (1998) 156, 275–282  1998 Journal of Endocrinology Ltd Printed in Great Britain 0022–0795/98/0156–0275 $08.00/0 Downloaded from Bioscientifica.com at 06/03/2022 05:03:11PM via free access