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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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.
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.
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.