Bong, Ji-Hong Song, Hyun-Woo Kim, Tae-Hun Kang, Min-Jung Jose, Joachim Pyun, Jae-Chul 2024-01-19T23:01:42Z 2024-01-19T23:01:42Z 2021-09-03 2018-04-15 0956-5663 https://pubs.kist.re.kr/handle/201004/121474 In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia colt 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. English ELSEVIER ADVANCED TECHNOLOGY ESCHERICHIA-COLI-CELLS OUTER-MEMBRANE LAYER LIGAND-BINDING ASSAYS Z-DOMAINS E. COLI SPR BIOSENSOR VARIABLE DOMAINS IMMUNOASSAY PROTEIN STABILITY Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors Article 10.1016/j.bios.2017.12.009 1 BIOSENSORS & BIOELECTRONICS, v.102, pp.600 - 609 BIOSENSORS & BIOELECTRONICS 102 600 609 scie scopus 000424176600079 2-s2.0-85038001290 Biophysics Biotechnology & Applied Microbiology Chemistry, Analytical Electrochemistry Nanoscience & Nanotechnology Biophysics Biotechnology & Applied Microbiology Chemistry Electrochemistry Science & Technology - Other Topics Article ESCHERICHIA-COLI-CELLS OUTER-MEMBRANE LAYER LIGAND-BINDING ASSAYS Z-DOMAINS E. COLI SPR BIOSENSOR VARIABLE DOMAINS IMMUNOASSAY PROTEIN STABILITY Negative regulatory factor (NEF) Autodisplay Single-chain variable fragment (scFv) Glutathione Immunoassay