title: Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity.
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.
authors.affiliation: School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China; E-Mails: jxzhao@jiangnan.edu.cn (J.-X.Z.); yanglian27@yahoo.com.cn (L.Y.); haiqinchen@jiangnan.edu.cn (H.-Q.C.); fwtian@jiangnan.edu.cn (F.-W.T.).
authors.forename: Jian-Xin
authors.initials: JX
authors.lastname: Zhao
authors.forename: Lian
authors.initials: L
authors.lastname: Yang
authors.forename: Zhen-Nan
authors.initials: ZN
authors.lastname: Gu
authors.forename: Hai-Qin
authors.initials: HQ
authors.lastname: Chen
authors.forename: Feng-Wei
authors.initials: FW
authors.lastname: Tian
authors.forename: Yong-Quan
authors.initials: YQ
authors.lastname: Chen
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000915:Antibody Affinity; D004220:Disulfides / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D004220:Disulfides; D057127:Single-Chain Antibodies