Real-time kinetic analysis of hCG–monoclonal antibody interaction using radiolabeled hCG probe: presence of two forms of Ag–mAb complex as revealed by solid phase dissociation studies

• Published in: Biochimica et biophysica acta Vol. 1569; no. 1; pp. 21 - 30
• Main Authors: Banerjee, Ashish, Srilatha, Nonavinakere Seetharam, Murthy, Gundlupet Satyanarayana
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15-01-2002
• Subjects: Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antigen-Antibody Complex - chemistry; Antigen-Antibody Reactions; Antigen–antibody; Chorionic Gonadotropin - immunology; Collodion; Dissociation; Human chorionic gonadotropin; Humans; Iodine Radioisotopes; Kinetics; Models, Theoretical; Protein Isoforms - chemistry; Real time kinetics; NC com, Antigen–monoclonal antibody complex on nitrocellulose; hLH, human luteinizing hormone; Antigen–antibody; mAb, monoclonal antibody; TBP, tight-binding portion; IhCG, 125I-labeled human chorionic gonadotropin; Real time kinetics; hFSH, human follicle-stimulating hormone; [ 125I]mAb, radio-iodinated monoclonal antibody; Human chorionic gonadotropin; RIA, radioimmunoassay; AH-sepharose, aminohexyl Sepharose; Dissociation; SPR, surface plasmon resonance; IBS, independent binding site model
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(01)00228-8

Real-time kinetics of ligand–ligate interaction has predominantly been studied by either fluorescence or surface plasmon resonance based methods. Almost all such studies are based on association between the ligand and the ligate. This paper reports our analysis of dissociation data of monoclonal antibody-antigen (hCG) system using radio-iodinated hCG as a probe and nitrocellulose as a solid support to immobilize mAb. The data was analyzed quantitatively for a one-step and a two-step model. The data fits well into the two-step model. We also found that a fraction of what is bound is non-dissociable (tight-binding portion (TBP)). The TBP was neither an artifact of immobilization nor does it interfere with analysis. It was present when the reaction was carried out in homogeneous solution in liquid phase. The rate constants obtained from the two methods were comparable. The work reported here shows that real-time kinetics of other ligand–ligate interaction can be studied using nitrocellulose as a solid support.