Dl. Baly et al., DEVELOPMENT OF A SPECIFIC AND SENSITIVE 2-SITE ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR MEASUREMENT OF INHIBIN-A IN SERUM, Endocrinology, 132(5), 1993, pp. 2099-2108
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.