Journal of Biosciences

, Volume 30, Issue 3, pp 359–370 | Cite as

Epitope mapping from real time kinetic studies — Role of cross-linked disulphides and incidental interacting regions in affinity measurements: Study with human chorionic gonadotropin and monoclonal antibodies

  • Nonavinakere Seetharam Srilatha
  • P. Tamil Selvi
  • Gundlupet Satyanarayana Murthy


Real time kinetic studies were used to map conformational epitopes in human chorionic gonadotropin (hCG) for two monoclonal antibodies (MAbs). The epitopes were identified in the regions (α5-14 and α55-62). The association rate constant (k+1) was found to be altered by chemical modification of hCG, and the ionic strength of the reaction medium. Based on these changes, we propose the presence of additional interactions away from the epitope-paratope region in the hCG-MAb reaction. We have identified such incidental interacting regions (IIRs) in hCG to be the loop region α35-47 and α60-84. The IIRs contribute significantly towards theK A of the interaction. Therefore, in a macromolecular interaction of hCG and its MAb,K A is determined not only by epitopeparatope interaction but also by the interaction of the nonepitopic-nonparatopic IIRs. However, the specificity of the interaction resides exclusively with the epitope-paratope pair.


Association constant epitope mapping incidental interacting regions kinetics monoclonal antibody 

Abbreviations used




β-c-terminal peptide




human chorionic gonadotropin


human luteinising hormone


radiolabelled125I human chorionic gonadotropin


incidental interacting regions


dissociation constant


association constant


affinity constant


Monoclonal antibody


phenyl methyl sulphonyl fluoride




single step solid phase radioimmunoassay


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  1. Ashish B, Venkatesh N and Murthy G S 2002a Structure function analysis: Lessons from human chorionic gonadotropin;Indian J. Exp. Biol. 40 434–447Google Scholar
  2. Ashish B, Srilatha N S and Murthy G S 2002b Real time kinetic analysis of hCG-monoclonal antibody interaction using radiolabelled hCG probe: Presence of two forms of Ag-MAb complex as revealed by solid phase dissociation studies;Biochim. Biophys. Acta 1569 21–30Google Scholar
  3. Ashish B, Tamil Selvi P and Murthy G S 2002c Thermodynamics of hCG-monoclonal antibody interaction: an analysis of real time kinetics data obtained using radiolabelled hCG probe;Biochim. Biophys. Acta 1572 31–36PubMedGoogle Scholar
  4. Berger P, Bidart J M, Delves P S, Dirnhofer S, Hoermann R, Isaacs N, Jackson A, Klonisch T, Lapthorn A J, Lund T, Mann K, Roitt I, Schwarz S and Wick G 1996 Immunochemical mapping of gonadotropins;Mol. Cell Endocrinol. 123 33–43CrossRefGoogle Scholar
  5. Fraker P J and Speck J C 1978 Protein and cell membrane iodination with sparingly soluble chloramide 1, 3, 4, 6-tetrachloro-3, 6-diphenylglycouril;Biochem. Biophys. Res. Commun. 80 849–857PubMedCrossRefGoogle Scholar
  6. Jackson A M, Berger P, Pixley M, Klein C, Hsueh A J and Biome I1999 The biological action of choriogonadotropin is not dependent on the complete native quaternary interactions between the subunits;Mol. Endocrinol. 13 2175–2188PubMedCrossRefGoogle Scholar
  7. Kumar S and Nussinov R 2002 Relationship between ion pair geometries and electrostatic strengths in proteins;Biophy. J. 83 1595–1612Google Scholar
  8. Lapthorn A J, Harris D C, Littlejohn A, Lustbader J W, Canfield R E, Machin K J, Morgan F J and Isaacs N W 1994 Crystal Structure of human chorionic gonadotropin;Nature (London) 369 455–461CrossRefGoogle Scholar
  9. Li Y, Li H, Yang F, Smith-Gill S J and Mariuzza R A 2003 X-ray snap shots of the maturation of the antibody response to a protein antigen;Nat. Struct. Biol. 10 482–488PubMedCrossRefGoogle Scholar
  10. Lobel L, Pollak S, Wang S, Chaney M and Lustbader J W 1999 Expression and Characterization of recombinant β -subunit hCG homodimer;Endocrine 10 261–270PubMedGoogle Scholar
  11. Matzuk M M, Hsueh A J W, LaPolt P, Tsafriri A, Keene J L and Biome I 1990 The biological role of the carboxy-terminal extension of human chorionic gonadotropin;Endocrinology 126 376–383PubMedCrossRefGoogle Scholar
  12. Mohan S, Sinha N and Smith-Gill S J 2002 Differences in electrostatic properties at antibody-antigen binding sites: implications for specificity and cross-reactivity;Biophys. J. 83 2946–2968PubMedGoogle Scholar
  13. Mohan S, Sinha N and Smith-Gill S J 2003 Modeling the binding sites of anti-hen egg white lysozyme antibodies Hy-HEL-8 and Hy-HEL-26: an insight into the molecular basis of cross-reactivity and specificity;Biophys. J. 85 3221–3236PubMedCrossRefGoogle Scholar
  14. Murthy G S 1996 Real time kinetic analysis of antigen-anti-body interaction using solid phase binding: Transformation of hCG-monoclonal antibody complex;Curr. Sci. 71 981–988Google Scholar
  15. Murthy G S and Srilatha N S 1996 Mapping of assembled epitopes with microgram quantities of antigen: Identification of an epitope at the receptor binding region of hFSH;Curr. Sci. 70 1019–1022Google Scholar
  16. Murthy G S and Venkatesh N1996 Determination of kinetics of epitope paratope interaction based on solid phase binding: an inexpensive alternative to biospecific interaction analysis;J.Biosci. 21 641–651Google Scholar
  17. Purohit S, Shao K, Balasubramanian S V and Bahl O P 1997 Mutants of human chorionic gonadotropin lacking N-glycosyl chain in the β-subunit.1.Mechanism for the differential action of the N-linked carbohydrates;Biochemistry 36 12355–12363PubMedCrossRefGoogle Scholar
  18. Sinha N, Mohan S, Lipschultz C A and Smith-Gill S J 2002 Electrostatics in protein binding and function;Curr. Protein Pept. Sci. 3 601–614PubMedCrossRefGoogle Scholar
  19. Sinha N and Smith Gill S J 2002 Protein structure to function via dynamics;Protein Pept. Lett. 9 367–377PubMedCrossRefGoogle Scholar
  20. Srilatha N S and Murthy G S 1996 Mapping of assembled epitopic regions of human chorionic gonadotropin reveals proximity of CTPα to the determinant loop β 93–100;J. Biosci. 21 755–764Google Scholar
  21. Srilatha N S and Murthy G S 2002 Study of dissociation of human chorionic gonadotropin monoclonal antibody complexes using nitrocellulose as an insoluble support;Curr. Sci. 78 1548–1552Google Scholar
  22. Tamil Selvi P, Ashish B and Murthy G S 2002 Determination of thermodynamic parameters of antigen-antibody interaction from real time kinetic studies;Curr. Sci. 82 1442–1448Google Scholar
  23. Troalen F, Bellet B H, Ghillani P, Puisieux A, Bohuon C J and Bidart J M 1988 Antigenic determinants on human chorionic gonadotropin a subunit. II. Immunochemical mapping by a monoclonal anti-peptide antibody;J. Biol. Chem. 263 10370–10376PubMedGoogle Scholar
  24. Venkatesh N, Nagaraja G and Murthy G S 1995 Analysis of a conformation specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes;Curr. Sci. 69 48–55Google Scholar
  25. Venkatesh N and Murthy G S 1997a Immunochemical approach to the mapping of an assembled epitope of human chorionic gonadotropin: Proximity of CTP-α to the receptor binding region of the B-subunit;J. Immunol. Methods 202 173–182PubMedCrossRefGoogle Scholar
  26. Venkatesh N and Murthy G S 1997b Stability of assembled epitopes of human chorionic gonadotropin to covalent modifications: Analysis using Monoclonal antibody probes;Biochem. Mol. Biol. Int. 42 853–863Google Scholar
  27. Venkatesh N, Krishnaswamy S, Meuris S and Murthy G S 1999 Epitope analysis and molecular modelling reveals the topography of the C-terminal peptide of β subunit of hCG;Eur. J. Biochem. 265 1061–1066PubMedCrossRefGoogle Scholar

Copyright information

© Indian Academy of Sciences 2005

Authors and Affiliations

  • Nonavinakere Seetharam Srilatha
    • 1
  • P. Tamil Selvi
    • 1
  • Gundlupet Satyanarayana Murthy
    • 1
  1. 1.Department of Molecular Reproduction, Development and GeneticsIndian Institute of ScienceBangaloreIndia

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