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

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

doi:10.1007/s00216-010-3535-9

Cited by 19 publications

(18 citation statements)

References 29 publications

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“…10 The previous research supposed that the highly sensitive fluorescence of AcGFP detected by the fluorescent microplate reader contributed to the improvement in sensitivity and this is consistent with our results. 21,22 The cross-reactivity values for other AVMs in the optimized FLISA were all consistent with that in traditional colorimetric ELISA method, while lower IC 50 in the FLISA was obtained (table 2). All these data showed that a one-step FLISA could be developed by using fluobody instead of monoclonal antibody.…”

Section: Comparison Of the Developed Flisa And Traditional Elisasupporting

confidence: 81%

Development of a fluorescence-linked immunosorbent assay for detection of avermectins using a fluorescent single-domain antibody

et al. 2015

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Abstract：：：：A fluorescent single-domain antibody (fluobody), consisting of a single-chain variable fragment antibody (scFv) and a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), was produced and used to develop a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) for avermectins (AVMs). The scFv gene was prepared by cloning V H and V L genes from hybridoma cell line 2C11 and then fused to the C-terminus of AcGFP (fluobody) with a flexible peptide linker (Gly 4 Ser) 2 between the two domains. After expression and purification, the functional fluobody was used to develop a one-step FLISA protocol for determination of AVMs in milk samples. The 50% inhibition concentration (IC 50 ) values and the limit of detection (LOD) of the optimized immunoassay for abamectin (ABM) were 2.13 and 1.07 ng mL -1 , respectively. Cross reactivity studies showed that the fluobody exhibited high affinity to the other four AVMs. The recoveries from the spiked milk samples ranged between 86.8 and 125.0%, with relative standard deviation lower than 10.2%. Moreover, the developed FLISA was applied to field samples, followed by confirmation with liquid chromatography-fluorescence detection (LC-FLD) analysis.The consistency of results between the immunoassay and instrumental techniques in detecting the presence of AVMs near the detection limit of the FLISA indicated that the newly developed method is suitable for rapid screening of AVMs contamination in food samples prior to chromatographic analysis.

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Section: Comparison Of the Developed Flisa And Traditional Elisasupporting

confidence: 81%

Development of a fluorescence-linked immunosorbent assay for detection of avermectins using a fluorescent single-domain antibody

et al. 2015

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“…In addition, sensitive FLISA can be performed by taking advantage of the strong fluorescence intensity of GFP [48]. Up to date, ic-FLISA can be developed to detect/determine small molecules, which include picloram [49], s-triazine [50], 5-methyl 2'-deoxycytidine [51] and bioactive natural product, plumbagin (PL) and ginsenoside Re (GRe) as previously described in our group [52]- [54]. The icFLISA for PL and GRe shows a lower limit of determination than conventional icELISA.…”

Section: Fluorescence-linked Immunosorbent Assaymentioning

confidence: 99%

Application of Green Fluorescent Protein in Immunoassays

Sakamoto¹,

Shoyama²,

Tanaka³

et al. 2014

ABB

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Green fluorescent protein (GFP) is a protein that emits green fluorescence when exposed to a radiation of ultraviolet wavelength range, even without the addition of substrate and cofactor. Because of such characteristics, the usage of GFP is widespread in both in vivo and in vitro applications. In addition, recent advances in biotechnology have enabled GFP to be expressed in various hosts, including bacteria, yeast, plants, animals, and even living-cells, for multiple purposes. Currently, GFP is a subject of great interest in the analytical sciences, especially in immunoassays for qualitative and quantitative analyses, when it is fused with an antibody because of the high sensitivity of GFP and antigen-binding specificity of antibodies. Recently the fluobody, which is a fusion protein of GFP with single-chain variable fragment antibody (scFv), has become a useful tool in various fields. We review here the applications of GFP as fluobodies in immunoassays.

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“…Currently, the usage of fluobody has been expanded from diagnosis, molecular targeted therapy, immunolabeling of cancer cells, probes for immunoassay, fluorescence-activated cell sorter (FACS) to fluorescent-linked immunosorbent assay (FLISA) for the detection of large molecular weight antigens such as proteins, peptides, and microtubules [47][48][49][50][51]. So far the application of a fluobody in FLISA for the detection of small molecules has been rarely reported except against herbicide, picloram [52] and s-triazine [53], and bioactive natural products, PL and G-Re [54][55][56] previously described by our group.…”

Section: Introductionmentioning

confidence: 99%

Fluobodies against Bioactive Natural Products and their Application in Fluorescence-Linked Immunosorbent Assay

et al. 2012

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An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb), Fab antibody, and single-chain variable fragment (scFv) antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP) with a scFv antibody, named fluobody, was proposed as a probe for an alternative immunosorbent assay; i.e., fluorescence-linked immunosorbent assay (FLISA). In this FLISA, an even more sensitive, simple, and rapid immunoassay can be performed by detecting the highly sensitive fluorophore of GFP that is genetically and directly fused to the scFv antibody. In addition, the time- and cost-consuming secondary antibody reaction and the following enzyme-substrate reaction, necessary for conventional ELISA, can be avoided, making it possible to complete the assay more rapidly. Focusing on naturally occurring bioactive products, fluobody recognizing 1,4-naphthoquinone, plumbagin and triterpenoid saponin, ginsenosides were successfully expressed in Escherichia coli (E. coli) and applied to FLISA. The construction, the expression, and the potential use of fluobody in quantitative/qualitative analysis of bioactive natural products are reviewed in this article

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

Cited by 19 publications

References 29 publications

Development of a fluorescence-linked immunosorbent assay for detection of avermectins using a fluorescent single-domain antibody

Development of a fluorescence-linked immunosorbent assay for detection of avermectins using a fluorescent single-domain antibody

Application of Green Fluorescent Protein in Immunoassays

Fluobodies against Bioactive Natural Products and their Application in Fluorescence-Linked Immunosorbent Assay

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