In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity.
Bibliographical noteFunding Information:
This work was supported by the Nano-Convergence Foundation [grant number: R201602210 ] funded by the Ministry of Science, ICT and Future Planning (MSIP, Korea) and Ministry of Trade, Industry and Energy (MOTIE, Korea), Industry Technology Development Program [grant number: 10063335 ] funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea) , and National Research Foundation of Korea [grant numbers: NRF-2017R1A2B4004077, NRF-2017R1A2B2004398 ].
All Science Journal Classification (ASJC) codes
- Biomedical Engineering