Antibody Recognition of a Human Chorionic Gonadotropin Epitope (hCGβ66–80) Depends on Local Structure Retained in the Free Peptide

Craig R Gregor,Eleonora Cerasoli,James Schouten,Jascindra Ravi,Jerry Slootstra,Adrian Horgan,Glenn J Martyna,Maxim G Ryadnov,Paul Davis,Jason Crain

doi:10.1074/jbc.m111.246637

Abstract

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical β3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGβ(66-80) and the closely related luteinizing hormone (LH) fragment LHβ(86-100), which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein.

Highlights

The role of Human chorionic gonadotropin (hCG) as a tumor marker in a variety of cancers is of great clinical significance [3, 4], and quantitative, specific hCG tests are important in the diagnosis and management of hCGsecreting cancers [5]
The sequence of the ␤3-loop is highly conserved with hCG␤66 – 80 and luteinizing hormone (LH)␤86 –100 differing by only one amino acid; asparagine (Asn) in hCG cf. aspartate (Asp) in LH (Fig. 2a)
We found that this single amino acid difference between LH and hCG confers a substantial specificity bias to the antigenantibody interaction (Fig. 2), in favor of hCG with all five of the monoclonal antibodies binding to this region

Summary

Introduction

The role of hCG as a tumor marker in a variety of cancers is of great clinical significance [3, 4], and quantitative, specific hCG tests are important in the diagnosis and management of hCGsecreting cancers [5]. Both the ␣- and ␤-chains are composed as three loops held in place by a “cysteine knot” of three disulfide bonds [1, 12] These similarities in sequence and structure, especially within the quartet of the glycoprotein protein hormones (hCG, LH, TSH, FSH), make stringent demands of antibody specificity when immunoassays are used to distinguish among the different molecules. The performance of a single site assay depends on the identity and nature of the chosen epitope, and it is crucial that it is present, intact and correctly folded in every hCG variant encountered It is against this background that the ␤3-loop of hCG␤66–80 (Fig. 1b) has been identified as a potentially ideal epitope on which to base universal, single site hCG assays. It is suggested here that this local structure provides the basis for antibodies against this epitope to bind with similar affinity to any known hCG variant, as long as the relevant linear sequence remains accessible

Methods

Results

Conclusion