Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin.
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.
Sakamoto
Seiichi
S
Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Taura
Futoshi
F
Pongkitwitoon
Benyakan
B
Putalun
Waraporn
W
Tsuchihashi
Ryota
R
Kinjo
Junei
J
Tanaka
Hiroyuki
H
Morimoto
Satoshi
S
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
D007164:Immunosorbents; D009285:Naphthoquinones; D057127:Single-Chain Antibodies; C014758:plumbagin