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

1 School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China 2 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China 3 Department of Cancer Biology, School of Medicine, Wake Forest University, Winston Salem, NC 27109, USA
* Author to whom correspondence should be addressed.
Received: 12 October 2010; in revised form: 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

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MDPI and ACS Style

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.

AMA Style

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. International Journal of Molecular Sciences. 2011; 12(1):1-11.

Chicago/Turabian Style

Zhao, Jian-Xin; Yang, Lian; Gu, Zhen-Nan; Chen, Hai-Qin; Tian, Feng-Wei; Chen, Yong-Quan; Zhang, Hao; Chen, Wei. 2011. "Stabilization of the Single-Chain Fragment Variable by an Interdomain Disulfide Bond and Its Effect on Antibody Affinity." Int. J. Mol. Sci. 12, no. 1: 1-11.

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