Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin

Seiichi Sakamoto, Futoshi Taura, Benyakan Pongkitwitoon, Waraporn Putalun, Ryota Tsuchihashi, Junei Kinjo, Hiroyuki Tanaka, Satoshi Morimoto

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4- naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly 4Ser)2 between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL -1) was improved to eightfold higher than that in conventional ELISA (0.2 μg mL-1), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.

Original languageEnglish
Pages (from-to)2955-2963
Number of pages9
JournalAnalytical and Bioanalytical Chemistry
Volume396
Issue number8
DOIs
Publication statusPublished - Apr 1 2010

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry

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