Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity

Int J Mol Sci. 2010 Dec 23;12(1):1-11. doi: 10.3390/ijms12010001.

Abstract

The interdomain instability of single-chain fragment variable (scFv) might result in intermolecular aggregation and loss of function. In the present study, we stabilized H4-an anti-aflatoxin B(1) (AFB(1)) scFv-with an interdomain disulfide bond and studied the effect of the disulfide bond on antibody affinity. With homology modeling and molecular docking, we designed a scFv containing an interdomain disulfide bond between the residues H44 and L100. The stability of scFv (H4) increased from a GdnHCl(50) of 2.4 M to 4.2 M after addition of the H44-L100 disulfide bond. Size exclusion chromatography revealed that the scFv (H44-L100) mutant existed primarily as a monomer, and no aggregates were detected. An affinity assay indicated that scFv (H4) and the scFv (H44-L100) mutant had similar IC(50) values and affinity to AFB(1). Our results indicate that interdomain disulfide bonds could stabilize scFv without affecting affinity.

Keywords: affinity; aflatoxin; disulfide bond; single-chain fragment variable; stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibody Affinity*
  • Disulfides / chemistry
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli / genetics
  • Models, Molecular
  • Protein Stability
  • Protein Structure, Tertiary
  • Single-Chain Antibodies / chemistry*

Substances

  • Disulfides
  • Single-Chain Antibodies