Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen |
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Authors: | Ashutosh Tiwari Durgashree Dutta Navin Khanna Subrat K Acharya Subrata Sinha |
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Institution: | (1) Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India;(2) International Center for Genetic Engineering and Biotechnology, New Delhi, India;(3) Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India |
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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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Keywords: | Fab fragment HBsAg Humanization Anti-HBs antibody Phage display |
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