Monoclonal and Polyclonal Antibodies Reactive with the 1-32 Amino Terminal Sequence of the Alpha Subunit of Human 32K Inhibin

Abstract

A monoclonal antibody was made after immunization of mice with the 1-32 amino terminal peptide of the alpha subunit of 32K human ovarian inhibin. The IgG2a mouse antibody reacted 6 times better with bovine 1-32 peptide than it did with 32K bovine inhibin. By contrast sheep polyclonal antibodies made by a similar method had a 29 fold bias in reactivity towards the immunizing peptide. Relative to homologous 1-32 peptide standards, the monoclonal antibody measured apparently higher amounts of immunoreactive material(s) in human (13.5 fold) and bovine (27 fold) follicular fluids than did the polyclonal anti 1-32 peptide antibodies. Immunochemical studies revealed that the epitope recognized by the monoclonal antibody was different from the major epitope recognized by the polyclonal antibodies. The monoclonal antibody reacted much better with human inhibin 1-32 sequences than with bovine (73 fold) or porcine (23 fold). Although the 32K form of human inhibin has not yet been purified, it can be inferred that the monoclonal antibody would be able to detect as little as 2 ng/ml of 32K human inhibin in competitive radioimmunoassays. The antibody must also react with some of the multiple molecular forms of inhibin found in human follicular fluids, and it was shown to function well in the quantitative immunoaffinity extraction of inhibin-like immunoreactivity from follicular fluid. It seems likely that this monoclonal antibody will prove a useful tool for research on human inhibin.

Regulation of immunoreactive inhibin A and B secretion in cultured human granulosa-luteal cells by gonadotropins, activin A and insulin-like growth factor type-1 receptor

“…8-BrcAMP was purchased from Sigma, staurosporine from Boehringer Mannheim (Mannheim, Germany), and IGF type-1 receptor antibody IR3 from Oncogene Sciences (Uniondale, NY, USA). Inhibin A and B were measured by specific enzymelinked immunosorbent assay (ELISA) kits (product codes MCA950 KZZ and MCA1312 KZZ respectively; Serotec Ltd, Oxford, Oxon, UK) as described previously (Groome et al 1990, Groome & Lawrence 1991. The detection limit for the assay was reported to be 2 pg/ml for inhibin A and 15 pg/ml for inhibin B.…”

The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: Characterization of the purified untagged processed mature region

“…The animals were killed and the spleens removed for fusion to Sp2/0 murine myeloma cells, following standard procedures. The hybridoma supernatants were initially screened by ELISA against the recombinant hBMP15 coated to Nunc immunoplates by standard protocol (Groome et al 1990). Reactive clones were expanded and recloned by limiting dilution.…”

Photoperiod-dependent regulation of inhibin in Siberian hamsters: I. Ovarian inhibin production and secretion

“…The current inhibin ELISAs use monoclonal antibodies against amino acids 1-32 of the human C region of the -subunit (Groome et al 1990). A comparison of the 1-32 amino acid sequence of the human and Siberian hamster mature -subunit reveals that there are four different amino acids which may lead to a reduced immunoactivity in the inhibin A and inhibin B ELISAs.…”