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Int. J. Mol. Sci. 2011, 12(1), 1-11; doi:10.3390/ijms12010001
Article

Stabilization of the Single-Chain Fragment Variable by an Interdomain Disulfide Bond and Its Effect on Antibody Affinity

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Received: 12 October 2010 / Revised: 8 December 2010 / Accepted: 16 December 2010 / Published: 23 December 2010
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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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 B1 (AFB1) 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 GdnHCl50 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 IC50 values and affinity to AFB1. Our results indicate that interdomain disulfide bonds could stabilize scFv without affecting affinity.
Keywords: aflatoxin; affinity; disulfide bond; single-chain fragment variable; stability aflatoxin; affinity; disulfide bond; single-chain fragment variable; stability
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Zhao, J.-X.; Yang, L.; Gu, Z.-N.; Chen, H.-Q.; Tian, F.-W.; Chen, Y.-Q.; Zhang, H.; Chen, W. Stabilization of the Single-Chain Fragment Variable by an Interdomain Disulfide Bond and Its Effect on Antibody Affinity. Int. J. Mol. Sci. 2011, 12, 1-11.

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