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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
Article

Abstract

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.

Keywords

Association constant epitope mapping incidental interacting regions kinetics monoclonal antibody 

Abbreviations used

Ac

Acetyl

β-BCTP

β-c-terminal peptide

Chy

chymotrypsin

hCG

human chorionic gonadotropin

hLH

human luteinising hormone

IhCG

radiolabelled125I human chorionic gonadotropin

IIR

incidental interacting regions

k−1

dissociation constant

k+1

association constant

KA

affinity constant

MAb

Monoclonal antibody

PMSF

phenyl methyl sulphonyl fluoride

Try

trypsin

SS-SPRIA

single step solid phase radioimmunoassay

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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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