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

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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 (Gly4Ser)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.

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Acknowledgments

This work was funded by the Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. The research in this paper was also supported, in part, by a Grant in Aid from the Japan Society for the Promotion of Science Asian CORE Program; the Ministry of Education, Culture, Sports, Science, and Technology of Japan; and the National Center for Genetic and Biotechnology (BIOTEC), Thailand

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Authors and Affiliations

  1. Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Seiichi Sakamoto, Futoshi Taura, Benyakan Pongkitwitoon, Hiroyuki Tanaka & Satoshi Morimoto

  2. Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand

    Waraporn Putalun

  3. Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka, 814-0180, Japan

    Ryota Tsuchihashi & Junei Kinjo

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  1. Seiichi Sakamoto
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  2. Futoshi Taura
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  3. Benyakan Pongkitwitoon
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  4. Waraporn Putalun
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  5. Ryota Tsuchihashi
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  7. Hiroyuki Tanaka
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  8. Satoshi Morimoto
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Correspondence to Hiroyuki Tanaka.

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Sakamoto, S., Taura, F., Pongkitwitoon, B. et al. Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin. Anal Bioanal Chem 396, 2955–2963 (2010). https://doi.org/10.1007/s00216-010-3535-9

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  • DOI: https://doi.org/10.1007/s00216-010-3535-9

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