Molecular Biotechnology

, Volume 43, Issue 1, pp 29–40

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

  • Ashutosh Tiwari
  • Durgashree Dutta
  • Navin Khanna
  • Subrat K. Acharya
  • Subrata Sinha
Research

DOI: 10.1007/s12033-009-9165-9

Cite this article as:
Tiwari, A., Dutta, D., Khanna, N. et al. Mol Biotechnol (2009) 43: 29. doi:10.1007/s12033-009-9165-9
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Abstract

5S is a mouse monoclonal IgG1 that binds to the ‘a’ epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (KD = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.

Keywords

Fab fragmentHBsAgHumanizationAnti-HBs antibodyPhage display

Copyright information

© Humana Press 2009

Authors and Affiliations

  • Ashutosh Tiwari
    • 1
  • Durgashree Dutta
    • 1
  • Navin Khanna
    • 2
  • Subrat K. Acharya
    • 3
  • Subrata Sinha
    • 1
  1. 1.Department of BiochemistryAll India Institute of Medical SciencesNew DelhiIndia
  2. 2.International Center for Genetic Engineering and BiotechnologyNew DelhiIndia
  3. 3.Department of GastroenterologyAll India Institute of Medical SciencesNew DelhiIndia