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

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 ␤3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCG␤ 66 -80 and the closely related luteinizing hormone (LH) fragment LH␤ 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.The glycoprotein human chorionic gonadotropin (hCG), 2 is a heterodimer consisting of an ␣-and ␤-chain (Fig. 1a) in noncovalent association. Essential in pregnancy and detectable within a few days of fertilization, it has become one of the most frequently assayed hormones, and simple, one-step, antibodybased measurements of hCG are now standard in pregnancy testing. At this early stage of pregnancy, the trophoblast cells of the preimplantation embryo produce a hyperglycosylated form of the hormone, which drives embryonic implantation in the uterine wall (2). Subsequently, hCG regulation by syncytiotrophoblast cells of the placenta promotes and maintains progesterone secretion from the corpus luteum. In other applications, therapeutically injected hCG is the key to in vitro fertilization, where it is used to trigger ovulation and, in males, used to promote testosterone production.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). However, the design criteria of hCG cancer assays differ from those required for pregnancy tests. Specifically, different sensitivity ranges are involved, and, for oncology applications, the antibodies used must be able to detect various modified forms of hCG with equal affinity (6). The role of hCG assays in the diagnosis and management of malignant trophoblastic disease (choriocarcinoma) is one of the great success stories of oncology (7,8); it is the ideal tumor marker, always present when choriocarcinoma cells exist, and in quantities directly related to the number of those cells. The correct use of hCG assays in combination with appropriate therapy has led to a survival rate approaching 100% in this otherwise aggressive cancer.All hCG diagnostic tests are carried out as immunoassays, based on specific antibodies that are able to distinguish hCG from three other, closely related, members of the glycoprotein hormone family (luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH)) (9). The ␣-chains of these four hormones are identica...

A Point-of-Care Immunosensor for Human Chorionic Gonadotropin in Clinical Urine Samples Using a Cuneated Polysilicon Nanogap Lab-on-Chip

“…Subsequently, the remaining free surface amine groups on the PSNG electrode after Ab immobilization were blocked with 5% BSA in PBS for 10 min to avoid non-specific binding of hCG on the electrode. Then, the–NH 2 protected by tBOC was removed by immersing the electrode in 10 mM TFA solution prior to detection with the hCG target [ 48 ].…”

Reconstructing the Discontinuous and Conformational β1/β3‐Loop Binding Site on hFSH/hCG by Using Highly Constrained Multicyclic Peptides

“…It is therefore likely that the presence of the b1-loop sequence, when long enough, will significantly strengthen binding of a single b3 loop to mAb 3468. For mAbs 4F9 and 8G5, previous binding experiments with linear b3-loop peptides in ELISA provided clear evidence for a b3-loop epitope, [37] but information other than that was not available. We observed that all three mAbs showed strong binding to tricyclic hCG mimic IVa by ELISA (Figure 3), virtually identical to that of hCG (less than twofold difference).…”