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

Prasad, Pramod V and Chaube, Shail K and Panchal, Manoj and Chaudhary, Rajesh and Muralidhar, K and Rohil, Vishwajeet and Kumari, GL and Kumar, Anand and Ashish, B and Murthy, GS and Shrivastav, TG (2007) Molecular dissection of an hCG-β epitope using single-step solid phase radioimmunoassa. In: Clinica Chimica Acta, 376 (1-2). pp. 52-59.

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Abstract

Background: 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. Method: 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) $G_1G_{10.}1$. The extent of competitive inhibition in binding ability of $^{125}IhCG-β$ with chemically or enzymatically modified hCG-β to immobilized MAb $G_1G_{10.}1$ in comparison to hCG-β standards was utilized to identify the epitopic amino acid involved in epitope–paratope interaction. Results: 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. Conclusion: 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.

Item Type:	Journal Article
Publication:	Clinica Chimica Acta
Publisher:	Elsevier
Additional Information:	Copyright of this article belongs to Elsevier.
Keywords:	Conformation-specific or assembled epitope;hCG;SS-SPRIA;MAb G1G10.1
Department/Centre:	Division of Biological Sciences > Molecular Reproduction, Development & Genetics
Date Deposited:	21 Feb 2007
Last Modified:	19 Sep 2010 04:35
URI:	http://eprints.iisc.ac.in/id/eprint/9940

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