reparation and Characterization of an Improved β-COOH-Terminal Immunogen for Generation of Specific and Sensitive Antisera to Human Chorionic Gonadotropin*

Antisera raised against the entire β-subunit of hCG usually cross-react to some extent with human LH. While this degree of cross-reaction in serum assays can be relatively minor, human LH becomes a greater source of error when urine specimens are concentrated for measurement of very low levels of hCG secretion. To circumvent this problem, peptides from the unique β-COOH-terminal region can be employed as immunogens to elicit antisera of greater specificity for hCG, but, to date, the synthetic and desialylated peptides from this region have failed to produce antibodies with sufficient affinities for clinical application. Since the presence of sialic acid on these peptides could influence their conformation in native hCG, we have undertaken to study the immunochemistry of sialic acid-containing βCOOH-terminal peptides. These peptides were produced by tryptic digestion of the β-subunit and separated from the disulfide-rich core by gel filtration. After reduction and carboxymethylation to destroy any contaminants possessing conformational epitopes, further purification was accomplished by gel filtration. A peptide containing residues 115–145 of the β-subunit and possessing 5.7 mol sialic acid/mol peptide was isolated and conjugated to thyroglobulin. The conjugate was used as an immunogen to produce antisera in rabbits. Six of eight rabbits responded with detectable antibodies, and four produced affinities ranging from 6.5 × 109 to 1.35 × 1010. liters/M. Rabbits immunized with the peptide preparation that had not been subjected to the conjugation procedure did not respond with measurable antibody titers. All of the antisera bound a peptide containing residues 123–145 as well as the peptide used for immunization, but had significantly reduced affinity for a peptide of residues 115–141, showing that the binding site was near the COOH-terminus. These data indicate that this form of immunogen can give rise to affinities equal to or greater than those elicited by synthetic or desialylated forms of the hCG β- COOH-terminal region. The sensitivity and specificity of these antibodies for native hCG make them potentially useful for the measurement of hormone in urine concentrates.