Characterization and molecular modeling of a highly stable anti-Hepatitis B surface antigen scFv.
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.
Bose
Biplab
B
Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, PIN-110029, India.
Chugh
Dipti A
DA
Kala
Mrinalini
M
Acharya
Subrat K
SK
Khanna
Navin
N
Sinha
Subrata
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D001666:Binding Sites, Antibody; D006510:Hepatitis B Antibodies / Q000737:chemistry* / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv