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.
Highlights
Recombinant antibodies are used in research, diagnostic, and therapeutic applications
We studied the affinity and stability of a single-chain fragment variable (scFv) (H4) [6] against aflatoxin B1 (AFB1) and an interdomain disulfide bridge containing a mutation of H44-L100, which were expressed in functional form in Escherichia coli (E. coli)
We focused on scFv stabilization by an interdomain disulfide bond
Summary
Recombinant antibodies are used in research, diagnostic, and therapeutic applications. The relatively easy production of the single-chain fragment variable (scFv) antibody in bacteria and affinity maturation explain the increasing importance of scFv in many applications. Insufficient interface stability between the heavy and light chains of scFv fragments has often been suggested to be the main cause of irreversible scFv inactivation. An antibody with improved stability could be generated by increasing the favorable interactions at the interface between the VH and VL domains; such antibodies are difficult to create [3]. In addition to the linker between the VH and VL domains, an interdomain disulfide bond was designed to stabilize the scFv [4]. Few reports described the effect of the interdomain disulfide bond on antibody affinity [5]
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