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

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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.

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Acknowledgments

This work has been funded from research grants from the Department of Biotechnology, India to Subrata Sinha. Ashutosh Tiwari was recipient of junior and senior research fellowship from Council of Scientific and Industrial Research and Indian Council of Medical Research; India. We are thankful to Dr. Dennis R. Burton and The Scripps Research Institute, La Jolla, USA for providing the vector pCOM3H. We are thankful to Mathura Prasad, Satish and Jyoti for their technical and secretarial assistance.

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Authors and Affiliations

  1. Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India

    Ashutosh Tiwari, Durgashree Dutta & Subrata Sinha

  2. International Center for Genetic Engineering and Biotechnology, New Delhi, India

    Navin Khanna

  3. Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India

    Subrat K. Acharya

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  1. Ashutosh Tiwari
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  2. Durgashree Dutta
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Correspondence to Subrata Sinha.

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Tiwari, A., Dutta, D., Khanna, N. et al. Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen. Mol Biotechnol 43, 29–40 (2009). https://doi.org/10.1007/s12033-009-9165-9

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