DEVELOPMENT OF A SPECIFIC AND SENSITIVE 2-SITE ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR MEASUREMENT OF INHIBIN-A IN SERUM

A polyclonal chicken antiserum against purified 32 -kilodalton (kDa) r ecombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) aga inst either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9 A9) were used to develop three sensitive InhA enzyme-linked immunosorb ent assays (ELISAs). The sensitivity of an ELISA using affinity-purifi ed chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) average d 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 coe fficients of variation of 2-5% and 8-10%, respectively, for all assays . The ELISA formats did not cross-react with purified rh-ActA or recom binant human transforming growth factor-beta1 or detect any immunoreac tive proteins in medium conditioned by cell lines expressing rh-ActA o r recombinant human transforming growth factor-beta1. The Ck/Ck ELISA detected significant amount, of immunoreactivity in medium from cells expressing the free alpha-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 successf ully quantitated inhibin in sera from patients undergoing ovulation in duction and in rats (with or without sc administration of pregnant fem ale 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 quan titating inhibin-A in biological fluids, with little cross-reactivity to free alpha-chain or inhibin-B.