Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors

Ji Hong Bong, Hyun Woo Song, Tae Hun Kim, Min Jung Kang, Joachim Jose, Jae Chul Pyun

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)600-609
Number of pages10
JournalBiosensors and Bioelectronics
Volume102
DOIs
Publication statusPublished - 2018 Apr 15

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biophysics
  • Biomedical Engineering
  • Electrochemistry

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