Kinetic analysis of a human chorionic gonadotropin-beta epitope-paratope interaction.
Kinetics of protein-protein or ligand-ligate interaction has predominantly been studied by optical spectroscopy (particularly fluorescence) and surface plasmon resonance biosensors. Almost all such studies are based on association kinetics between ligand-ligate and suffer from certain methodological and interpretational limitations. Therefore, kinetic analyses of dissociation data of such interactions become indispensable. In the present investigation, the radiolabeled human chorionic gonadotropin-beta ((125)IhCGbeta) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G(1)G(10).1. The NC-G(1)G(10).1-(125)IhCGbeta complex (NC(com)) was prepared and the dissociation of radiolabeled hCGbeta was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k(- 1)), association constants (k(+1)) and affinity constants (k(a)) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCGbeta involved in interaction with the complementary paratope on MAb G(1)G(10).1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of hCGbeta epitope consists of Arg (94,95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Based on the results of present investigation, we conclude that dissociation kinetics coupled with SS-SPRIA unequivocally provides considerable insight into the study of ligand-ligate interactions and epitope analysis.
Prasad
Pramod Vishwanath
PV
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067, India. pkumar@popcouncil.org
Rani
Aruna
A
Chaube
Shail Kumar
SK
Rohil
Vishwajeet
V
Shrivastav
Tulsidas G
TG
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000936:Antigen-Antibody Complex; D000937:Antigen-Antibody Reactions / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology / Q000502:physiology*; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003101:Collodion; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D018997:Chorionic Gonadotropin, beta Subunit, Human; D000939:Epitopes; D007457:Iodine Radioisotopes; D008024:Ligands; D003101:Collodion