Novel assay for determination of androgen bioactivity in human serum

We have developed a mammalian cell(COS-l) bioassay, which can measure andro gen bioactivity directly from a small amount (10 muL) of human serum. The r ecombinant assay is based on androgen-dependent interaction between the lig and-binding domain and the N-terminal region of the androgen receptor (AR), which were fused to Ga14 DNA-binding domain of Saccharomyces cerevisiae an d transcriptional activation domain of herpes simplex VP16 protein, respect ively. The interaction is amplified by coexpression of AR-interacting prote in 3 in the cells. The reporter plasmid contains 5 Gal4-binding sites upstr eam of the luciferase gene; luciferase activity in cell lysates is derived from androgen bioactivity in human serum. Saturating concentration of testo sterone in FCS induced more than 700-fold induction in relative luciferase activity. The sensitivity was less than 1.0 nmol/L testosterone in FCS. The intra- and interassay coefficients of variation were 8.3% and 21%, respect ively. Interaction between the AR termini was blocked by nonsteroidal antia ndrogens, and the assay exhibited minimal cross-reactivity with 17 beta -es tradiol. Serum androgen bioactivity was studied in 23 boys (13.9-16.8 yr ol d) with constitutional delay of puberty and in 9 prepubertal boys with cryp torchidism (1.0-6.4 yr old). Androgen bioactivity was detectable in 15 boys with constitutional delay of puberty and in all boys with cryptorchidism d uring treatment with human CG (range, 1.0-14.5 nmol/L testosterone equivale nts). Serum androgen bioactivity measured by the bioassay correlated strong ly with serum testosterone concentration (r = 0.93, P < 0.0001, n = 22) but not to 5 alpha -dihydrotestosterone, dehydroepiandrosterone, or androstene dione levels. We conclude that our novel bioassay enables quantitation of m ammalian cell response to bioactive androgens in human serum, even in pedia tric patients with relatively low androgen levels.