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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.

SUBMITTER: Zhao JX 

PROVIDER: S-EPMC3039938 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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

Zhao Jian-Xin JX   Yang Lian L   Gu Zhen-Nan ZN   Chen Hai-Qin HQ   Tian Feng-Wei FW   Chen Yong-Quan YQ   Zhang Hao H   Chen Wei W  

International journal of molecular sciences 20101223 1


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 Gdn  ...[more]

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