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Deciphering a Conformation-Specific Epitope of hCG-Through Immunokinetics

Prasad, Pramod Vishwanath and Rani, Aruna and Chaube, Shail Kumar and Shrivastav, Tulsidas G (2009) Deciphering a Conformation-Specific Epitope of hCG-Through Immunokinetics. In: Journal of Immunoassay and Immunochemistry, 30 (01). pp. 1-17.

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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- (125IhCG) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G1G10.1. The NC-G1G10.1-125IhCG complex (NCcom) was prepared and the dissociation of radiolabeled hCG 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 (ka) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCG involved in interaction with the complementary paratope on MAb G1G10.1. 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 hCG 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 hCG

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Taylor and Francis.
Department/Centre: Division of Biological Sciences > Molecular Reproduction, Development & Genetics (formed by the merger of DBGL and CRBME)
Date Deposited: 11 Mar 2009 11:10
Last Modified: 19 Sep 2010 05:25
URI: http://eprints.iisc.ernet.in/id/eprint/18711

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