Synthetic Peptide Segments of Inhibin α- and β-Subunits: Preparation and Characterization of Polyclonal Antibodies*

Abstract

The amino acid sequences of human inhibin alpha-, beta A- and beta B-subunits were analyzed for hydrophilicity and chain flexibility to predict regions that are on the surface of the subunits and, therefore, are potential antigenic sites. Based on these analyses, a total of nine peptides were synthesized, and rabbit antisera against the peptides were prepared. Peptides of the N-terminus (residues 1-16 and 13-24) and region 109-123 of the alpha-subunit produced high titer antibodies. Regions 69-79 and 93-105 of the beta A-subunit and region 93-104 of the beta B-subunit were also immunogenic. Immunoblotting of an inhibin preparation with anti-alpha-peptide antiserum revealed that a 32K band (inhibin) and an 18K band (alpha-subunit) were stained. Immunoblotting with anti-beta-peptide antiserum detected a 32K band (inhibin), a 24K band (activin), and a 14K band (beta-subunit). Injection (iv) of these antisera into rats induced dramatic elevation of serum FSH in 6-12 h and suggested immunoneutralization of endogenous inhibin. RIAs for each subunit were developed using radioiodinated peptides as tracers. Competition binding assays indicated crossreactivity with human follicular fluid, semen, serum, plasma, and crude inhibin preparations. Parallel dilution curves were obtained. Antisera against beta A- and beta B-subunit peptide cross-reacted with each other. In immunocytochemical studies, these antisera were used in conjunction with gold-labeled goat antirabbit immunoglobulin G to localize inhibin in cells of the rat testis. Specific staining of inhibin was localized within the Sertoli cells of some tubules in adult rat testis. Positive staining could be blocked by preadsorbing the sera with the appropriate synthetic peptide. These results suggest that antibodies against synthetic inhibin peptides are useful in elucidating the roles of inhibin and activin.

Measurement of dimeric inhibin using a modified two-site immunoradiometric assay specific for oxidized (Met O) inhibin

“…Alternatively, it is possible that the clone E4 may have been selected for during hybridoma screening because the immobilized ß 82-114 peptide and/or native inhibin used to screen culture supernatants had oxidized spontaneously during the prep¬ aration and/or storage of antigen-coated plates. Several other groups (Saito et al 1989, Vaughan & Vale 1993 have reported the use of antibodies against C-terminal peptide sequences of inhibin ß subunit and it would be of interest to determine whether these antibodies also have a greater affinity for the oxidized antigen. If this was the case with the ßA81-113 antibody used by Vaughan & Vale (1993), it could account for the inability of their two-site IRMA to detect dimeric inhibin in crude human and porcine FF.…”

Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment

“…The hCG-treated cells expressed at least 11 additional polypeptides and four polypeptides decreased as the result of this treatment. By immunoprecipitation it was further determined that hCG stimulated the synthesis of a 14 kDa product corresponding to a and Ob inhibin subunits, respectively [32].…”

“…Samples containing equal amounts of trichloroacetic acid-precipitable radioactivity and approximately 50 pg of protein were diluted 1:lO with NET buffer (150 mM NaCl, 5 mM EDTA; 50 mM Tris-HC1, pH 7.5, containing 1.5% Nonidet P-40 and 2.5% mg/mL bovine serum albumin). After mixing, 2 pL of either undiluted rabbit anti-inhibin a or anti-inhibin fib antisera [32] were added; the mixture was incubated for 2 h at 4"C, then 30 pL of a 50% suspension of protein A-Sepharose were added; the samples were incubated for an additional 30 min at 4°C with rotation. The samples were then centrifuged at 12000 g, at 4"C, resuspended twice with brief sonication in NET buffer containing 0.1 O/ o Nonidet P-40, and pelleted as above.…”

Epitope Mapping of the Transforming Growth Factor-β Superfamily Protein, Macrophage Inhibitory Cytokine-1 (MIC-1): Identification of at Least Five Distinct Epitope Specificities

“…Most studies in which antibodies against TGF-β superfamily cytokines have been characterized have utilized antipeptide antibodies (5,13,(18)(19)(20)(21)(22)(23)(24), although some anti-protein monoclonal antibodies (MAbs) have also been described (25)(26)(27). The majority of the above reports have concentrated on determining the ability of the antiserum to neutralize biological activity of a particular cytokine or the suitability of the antiserum for use in cytokine-specific assays.…”