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1.
J Immunoassay Immunochem ; 30(1): 1-17, 2009.
Article in English | MEDLINE | ID: mdl-19117199

ABSTRACT

Proteins and peptides are comprised of both sequence-specific and conformation-specific epitopes. Sequence-specific epitopes are delineated by a peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays, etc. Available methods for deciphering conformation-specific epitopes are cumbersome (X-ray crystallography, etc.), time-consuming, and require expensive equipment. Therefore, it is indispensable to develop a simple method for identification and mapping of conformation-specific epitopes. 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 a conformation-specific epitope of hCGbeta consists of Arg (94, 95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Therefore, the results of the present investigation suggested that the dissociation kinetics coupled with SS-SPRIA unequivocally assists in deciphering amino acid residues constituting a conformation-specific epitope of hCGbeta.


Subject(s)
Chorionic Gonadotropin, beta Subunit, Human/immunology , Epitope Mapping/methods , Epitopes/immunology , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions/immunology , Binding Sites, Antibody/immunology , Chorionic Gonadotropin, beta Subunit, Human/chemistry , Epitopes/chemistry , Humans , Protein Conformation , Radioimmunoassay
2.
Growth Factors ; 26(6): 331-42, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19012067

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/metabolism , Antigen-Antibody Reactions , Binding Sites, Antibody/physiology , Chorionic Gonadotropin, beta Subunit, Human/metabolism , Epitope Mapping/methods , Epitopes/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex , Antigen-Antibody Reactions/immunology , Binding Sites, Antibody/immunology , Chorionic Gonadotropin, beta Subunit, Human/chemistry , Chorionic Gonadotropin, beta Subunit, Human/immunology , Collodion , Epitopes/chemistry , Epitopes/immunology , Humans , Iodine Radioisotopes , Kinetics , Ligands , Radioimmunoassay
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