Identification of Cross-Reactive and Restricted Epitopes Localized on Human Chorionic Gonadotropin Beta-Subunit by Monoclonal Antibodies

Abstract

Human chorionic gonadotropin (hCG) belongs to the family of glycoprotein hormones. All members of the family are composed of an identical α subunit and structurally related β subunit which confers biological specificity. Specific quantification and functional analysis of hCG require the use of monoclonal antibodies recognizing different epitopes of hCGβ. This study describes the production and characterization of monoclonal antibodies (MAbs) to hCGβ with no cross-reactivity to other glycoprotein hormones. Spleen cells from Balb/c mice immunized with hCG were fused with mouse SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of highly purified and recombinant glycoprotein hormones, their subunits and peptides representing the C-terminal end of hCGβ (hCGβ-CTP) by ELISA and immunoblotting. The affinity constant (K_aff) was also determined by ELISA. Three murine hybridomas designated G5M1, B12M2 and F4M3 were obtained that secrete MAbs specific for hCGβ. The G5M1 MAb reacts only with hCGβ, hCGβ-CTP and intact hCG with no detectable cross-reaction with hCGα or any of the other glycoprotein hormones. The specificity of B12M2 MAb is very similar to G5M1, but it does not react with hCGβ-CTP. The F4M3 MAb also has similar specificity to G5M1 and B12M2, but it strongly cross-reacts with hLH. The affinity constant (Kaff) of G5M1, B12M2 and F4M3 was found to be 4.28 × 10⁹, 5.2 × 10⁸, and 1.97 × 10⁹ M^-1, respectively. Our results indicate that G5M1 and B12M2 MAbs are specific for hCG and recognize epitopes restricted to hCGβ, but F4M3 recognizes a common epitope expressed both on hCGβ and hLHβ.