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.
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.
Banerjee
Ashish
A
Primate Research Laboratory, Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore 560 012, India.
Srilatha
Nonavinakere Seetharam
NS
Murthy
Gundlupet Satyanarayana
GS
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000936:Antigen-Antibody Complex / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D006063:Chorionic Gonadotropin / Q000276:immunology*; D003101:Collodion; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008962:Models, Theoretical; D020033:Protein Isoforms / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D006063:Chorionic Gonadotropin; D007457:Iodine Radioisotopes; D020033:Protein Isoforms; D003101:Collodion