Epitope mapping from real time kinetic studies - role of crosslinked disulphides and incidental interacting regions in affinity measurements: study with human chorionic gonadotropin and monoclonal antibodies.

Real time kinetic studies were used to map conformational epitopes in human chorionic gonadotropin (hCG) for two monoclonal antibodies (MAbs). The epitopes were identified in the regions (alpha 5--14 and alpha 55--62). The association rate constant (k+1) was found to be altered by chemical modification of hCG, and the ionic strength of the reaction medium. Based on these changes, we propose the presence of additional interactions away from the epitope- paratope region in the hCG-MAb reaction. We have identified such incidental interacting regions (IIRs) in hCG to be the loop region alpha 35--47 and alpha 60--84. The IIRs contribute significantly towards the KA of the interaction. Therefore, in a macromolecular interaction of hCG and its MAb, KA is determined not only by epitopeparatope interaction but also by the interaction of the nonepitopic-nonparatopic IIRs. However, the specificity of the interaction resides exclusively with the epitope-paratope pair.

[Production and characterization of a murine monoclonal antibody recognizing a conformational epitope on hCG]

Human chorionic gonadotropin hormone [hCG] belongs to glycoprotein hormones family. Other members of this family include follicle stimulating hormone [FSH], luteinizing hormone [LH] and thyroid stimulating hormone [TSH]. All these hormones consist of a common alfa and a distinct beta subunit. There is a strong similarity between the members of these hormones. Therefore, detection and quantitative measurment of these hormones require production of monoclonal antibodies specific for non-overlapping epitopes on the beta chain or a conformational epitope specific for each hormone. In this study a murine monoclonal antibody against the hCG dimer molecule was produced by hybridoma technology. The specificity of the antibody was assessed by ELISA and Immunoblotting using a panel of highly purified and recombinant forms of glycoprotein hormones including: native hCG and hLH, recombinant hCG, beta hCG, hCG alpha, beta hCG carboxyl terminal peptide covering amino acid residues 109-145 [beta hCG-CTP], recombinant TSH and native FSH, as well as urine proteins [UP]. It was found that the monoclonal antibody reacted with, dimer recombinant and urine purified hCG and hLH, but not with the reduced form of the hormone, nor with recombinant beta hCG, alpha hCG, TSH, native FSH and UP. Using beta hCG-CTP fragment with different concentrations to monitor inhibition of hormone- monoclonal antibody interactions, no interference was observed. This implies that the epitope recognized by the monoclonal antibody is different from that presented by beta hCG-CTP. These results suggest that the monoclonal antibody recognizes a conformational epitope located at the dimer form of hCG molecule and closely associated with the beta subunit of the hormone

Combined immunoprecipitation and agglutination for the detection of the heterodimeric molecule: human chorionic gonadotropin as a study model.

Double-particle reversed passive hemagglutination (RPHA) was performed for the detection of an intact heterodimeric form of human chorionic gonadotropin (intact hCG) composed in Profasi hCG (P-hCG). This technique relied on two mixed types of human O RBC, which were individually coated with two distinct monoclonal antibodies that recognized alpha or beta subunit of hCG, i.e. ALC-1 and BEL-5, respectively. The positive hemagglutination result was achieved by this technique. However, in the BEL-5 coated single-particle control system, positive results for both P-hCG and beta subunit hCG solution were realized. The occurrence of beta-multimer hCG was a causative molecule revealed by the hemagglutination inhibition technique. Thereby, the novel method called "combined immunoprecipitation and agglutination" was developed to overcome this problem. The free beta subunit together with the betamultimer hCG were eliminated from other forms presented in P-hCG after using the ALC-1 coated particles. The precipitated particles, which captured the heterodimer hCG molecule, reacted further with soluble BEL-5 to subsequently form a trellis. A positive result was obtained only with P-hCG, but not with beta subunit hCG or hLH. This study is inferable as a model for the detection of heterodimeric molecule by an elementary method.