Antibodies to the carboxyl-terminal fragment of human chorionic gonadotropin beta-subunit: characterization of antibody recognition sites using synthetic peptide analogues
Matsuura, S.; Chen, H.C.; Hodgen, G.D.
Biochemistry 17(4): 575-580
ISSN/ISBN: 0006-2960 PMID: 75021 DOI: 10.1021/bi00597a003
The immunochemical specificities of an antiserum (H-93) generated by immunization of a rabbit with a bovine serum albumin conjugate of a unique COOH[carboxyl]-terminal tricosaglycopeptide fragment (residues 123-145) isolated after tryptic digestion of the S-carboxymethylated, desialylated .beta.-subunit of human chorionic gonadotropin (hCG) were analyzed systematically with a radioimmunoassay system. Using [125I]hCG as labeled antigen, a series of 32 synthetic peptides of various lengths, analogous to the polypeptide sequence of the native antigen, was evaluated for antigenic recognition by the antiserum. Immunological cross-reactivity with the dipeptide, Pro-Gln, but not with Gln or Pro-Glu was observed. The degree of immunoreactivity increased with increasing chain length and reached a plateau at the pentadecapeptide, which was equipotent with a highly pruified hCG. The dose-response curves of hCG and the pentadecapeptide or longer synthetic peptides corresponding to the COOH-terminal peptide were superimposable. Thus, the antibody recognition was shown to reside at the last 15 amino acid residues of the COOH-terminal peptide of hCG.beta. Evidnece is also presented to indicate that a free COOH terminus is not essential for immunological cross-reactivity. Although cross-reactivity increased most significantly over the peptide sequence Arg-Leu-Pro-Gly (residues 133-136), 2 peptides containing this sequence, but lacking the last portion of COOH-terminal peptide, showed significantly lower (residues 125-137) or in significant (residues 131-137) cross-reactivity. The Pro-Gln dipeptide segment is 1 of the important recognition units for the antibody and the addition of the other 13 residues enhances the cross-reactivity by 5 .times. 104. Four more antisera produced in rabbits treated with the identical immunogen exhibited binding characteristics very similar to that of the H-93 antiserum. These studies describe the nature of the sites of antibody recognition and provide the basis for the high degree of specificity for hCG of the H-93 antiserum without cross-reactivity to structually similar hLH [luteinizing hormone].