Synthetic peptide segments of inhibin alpha- and beta-subunits: preparation and characterization of polyclonal antibodies.
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.
Saito
S
S
Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905.
Roche
P C
PC
McCormick
D J
DJ
Ryan
R J
RJ
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007265:Inhibins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D008297:Male; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D011863:Radioimmunoassay; D051381:Rats; D013737:Testis / Q000166:cytology / Q000378:metabolism
D000906:Antibodies; D010446:Peptide Fragments; D007265:Inhibins