title: Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin.
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(4)Ser)(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 microg mL(-1)), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Futoshi
authors.initials: F
authors.lastname: Taura
authors.forename: Benyakan
authors.initials: B
authors.lastname: Pongkitwitoon
authors.forename: Waraporn
authors.initials: W
authors.lastname: Putalun
authors.forename: Ryota
authors.initials: R
authors.lastname: Tsuchihashi
authors.forename: Junei
authors.initials: J
authors.lastname: Kinjo
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
mesh_terms: D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods*; D007164:Immunosorbents / Q000032:analysis*; D057230:Limit of Detection; D009285:Naphthoquinones / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D007164:Immunosorbents; D009285:Naphthoquinones; D057127:Single-Chain Antibodies; C014758:plumbagin