Development of a specific and sensitive two-site enzyme-linked immunosorbent assay for measurement of inhibin-a in serum

A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin- A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme- linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 ± 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 ± 6 pg/ml in a 10% serum matrix, with intra- and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-di or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-β. The Ck/Ck ELISA detected significant amounts of immunoreactivity in medium from cells expressing the free a-subunit of inhibin and recombinant inhibin-B (rh-InhB). In contrast, the mAb/Ck ELISAs showed no cross-reactivity to medium conditioned by these two cell lines. All three ELISA formats detected rh-InhA added to either human or rat serum in vitro or serum from rats injected with rh- InhA. The Ck/Ck and 9A9/Ck ELISAs successfully quantitated inhibin in sera from patients undergoing ovulation induction and in rats (with or without sc administration of pregnant female serum gonadotropin). The 11B5/Ck ELISA appeared to be specific for the 32-kDa form of inhibin, while the 9A9/Ck ELISA was useful in quantitating inhibin- A in biological fluids, with little cross-reactivity to free a-chain or inhibin-B.