Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 77, Issue 1, Pages 124-130Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2011.01.010
Keywords
Enzyme-linked immunosorbent assay; Fluobody; Fluorescence-linked immunosorbent assay; Ginsenosides; Green fluorescent protein; Single-chain variable fragment
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Funding
- Japan Society for the Promotion of Science for Young Scientists
- Japan Society for the Promotion of Science of the Ministry of Education, Culture, Sports, Science, and Technology of Japan
- Grants-in-Aid for Scientific Research [23810030] Funding Source: KAKEN
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A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv. (c) 2011 Elsevier Inc. All rights reserved.
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