Immunochemical mapping of a specific domain on human choriogonadotropin using anti-protein and anti-peptide monoclonal antibodies

• Published in: The Journal of biological chemistry 1987-11, Vol.262 (32), p.15483-15489
• Main Authors: Bidart, J M, Troalen, F, Bohuon, C J, Hennen, G, Bellet, D H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Antibodies, Monoclonal; Biological and medical sciences; Chorionic Gonadotropin - analysis; Chorionic Gonadotropin - metabolism; Epitopes - analysis; Fundamental and applied biological sciences. Psychology; Glycoprotein Hormones, alpha Subunit; Glycoproteins; Horses; Humans; Peptide Fragments - immunology; Pituitary Hormones, Anterior - analysis; Proteins; Receptors, LH - metabolism; Species Specificity; Swine
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)47752-6

In an attempt to localize topographic domains specific to native human chorionic gonadotropin (hCG), we studied the discontinuous antigenic regions recognized by a monoclonal anti-hCG antibody designated as C8 which binds only to hCG and does not cross-react with either the free hCG-alpha and hCG-beta subunits or other glycoprotein hormones. Using two-site monoclonal immunoradiometric assays (M-IRMAs), we found that C8 antibody and an anti-peptide antibody (FB12) directed to residues 110-116 of hCG-beta did not bind simultaneously to hCG. This observation suggested that C8 binds to residues of hCG-beta included either in the antibody-binding region of FB12 or in close proximity to amino acids 110-116. To further delineate the regions of hCG-beta recognized by C8, we carried out hapten inhibition experiments with synthetic peptides corresponding to various regions of hCG-beta. The peptide corresponding to residues 109-122 and subpeptides (111-122 or 112-122) inhibited the binding of 125I-hCG to C8, whereas weak inhibition was observed with subpeptide 113-122. By studying the binding of C8 to the 1-112 disulfide-bonded part of hCG-beta (hCG-beta core) recombined with hCG-alpha, we were able to confirm that C8 binds to a region including or near to Asp112. M-IRMAs showed that C8 does not bind to the recombinant molecule lacking residues 113-145 of hCG. Taken together, these results indicate that a limited number of residues located on hCG-beta near to Asp112, and most likely the sequence Asp111-Asp112-Pro113, are included in the discontinuous antigenic region recognized by C8. We then attempted to localize residues of hCG-alpha that constitute another part of the determinant which bound to C8. Six synthetic peptides corresponding to various regions of hCG-alpha did not inhibit binding of 125I-hCG to C8. In contrast, M-IRMAs demonstrated that C8 is capable of binding recombinant products composed of the hCG-beta subunit and the alpha subunits from human, equine, and porcine species. These results indicate that C8 recognizes a region of the alpha subunit highly conserved in these three species. Finally, we determined that the discontinuous regions recognized by C8 are partially accessible on the CG/LH-receptor complex.