Deciphering a conformation-specific epitope of hCG-beta through Immunokinetics.
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.
Prasad
Pramod Vishwanath
PV
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India. pkumar@popcouncil.org
Rani
Aruna
A
Chaube
Shail Kumar
SK
Shrivastav
Tulsidas G
TG
D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D011487:Protein Conformation; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D018997:Chorionic Gonadotropin, beta Subunit, Human; D000939:Epitopes