Characterization and molecular modelling of a highly stable anti hepatitis B surface antigen scFv

Bose, Biplab ; Chugh, Dipti A. ; Kala, Mrinalini ; Acharya, Subrat K. ; Khanna, Navin ; Sinha, Subrata (2003) Characterization and molecular modelling of a highly stable anti hepatitis B surface antigen scFv Molecular Immunology, 40 (9). 617- 631. ISSN 0161-5890

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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-antibody 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.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Anti- HBs Antibody; Recombinant Antibody; Protein-protein Interactions; Somatic Mutations; Molecular Modeling
ID Code:62584
Deposited On:22 Sep 2011 02:54
Last Modified:03 Jul 2012 07:52

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