Molecular dissection of an hCG-β epitope using single-step solid phase radioimmunoassay

Peptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time-consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. In the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of ‘mouse IgG-anti-mouse IgG’ was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) G1G10.1. The extent of competitive inhibition in binding ability of 125IhCG-β with chemically or enzymatically modified hCG-β to immobilized MAb G1G10.1 in comparison to hCG-β standards was utilized to identify the epitopic amino acid involved in epitope–paratope interaction. Data clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. The data of SS-SPRIA revealed the 93–100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-β at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.