Characterization and molecular modeling of a highly stable anti-Hepatitis B surface antigen scFv

被引:17
作者
Bose, B
Chugh, DA
Kala, M
Acharya, SK
Khanna, N
Sinha, S
机构
[1] All India Inst Med Sci, Dept Biochem, New Delhi 110029, India
[2] Int Ctr Genet Engn & Biotechnol, New Delhi, India
[3] All India Inst Med Sci, Dept Gastroenterol, New Delhi 110029, India
关键词
Anti-HBs antibody; recombinant antibody; protem-protein interactions; somatic mutations; molecular modeling;
D O I
10.1016/j.molimm.2003.07.002
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We raised a mouse monoclonal antibody (5S) against the 'a' epitope of the Hepatitis B surface antigen (HBsAg) by selecting for binding of the hybridoma supernatant in conditions that usually destabilize protein-protein interactions. This antibody, which was protective in an in vitro assay, had a high affinity with a relative dissociation constant in the nanomolar range. It also displayed stable binding to antigen in conditions that usually destabilize antigen-anti body interactions, like 30% DMSO, 8 M urea, 4 M NaCl, 1M guanidium HCl and extremes of pH. The variable regions of the antibody were cloned and expressed as an single chain variable fragment (scFv) (A5). A5 had a relative affinity comparable to the mouse monoclonal and showed antigen binding in presence of 20% DMSO, 8 M urea and 3 M NaCl. It bound the antigen in the pH range of 6-8, though its tolerance for guanidium HCl was reduced. Sequence analysis demonstrated a significant increase in the frequency of somatic replacement mutations in CDRs over framework regions in the light but not in the heavy chain. A comparison of the molecular models of the variable regions of the 5S antibody and its germ-line precurser revealed that critical mutations in the heavy and light chains interface resulted in better inter-chain packing and in the movement of CDR H3 and CDR L1 from their germline positions, which may be important for better antigen binding. In addition to providing a reagent for neutralizing for the virus, such an antibody provides a model for the evolution of stable high affinity interaction during antibody maturation. (C) 2003 Elsevier Ltd. All rights reserved.
引用
收藏
页码:617 / 631
页数:15
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