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

Mol Immunol. 2003 Dec;40(9):617-31. doi: 10.1016/j.molimm.2003.07.002.

Abstract

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, 1 M 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 precursor 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal
  • Antigen-Antibody Reactions
  • Base Sequence
  • Binding Sites, Antibody
  • Hepatitis B Antibodies / chemistry*
  • Hepatitis B Antibodies / genetics
  • Hepatitis B Surface Antigens / immunology*
  • Hybridomas
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Fragments / immunology*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Recombinant Proteins / immunology

Substances

  • Antibodies, Monoclonal
  • Hepatitis B Antibodies
  • Hepatitis B Surface Antigens
  • Immunoglobulin Fragments
  • Recombinant Proteins
  • immunoglobulin Fv