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Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides.

ABSTRACT: Phage-displayed peptides have been proven to be powerful reagents for competitive and noncompetitive immunoassays. However, they are unconventional reagents, which greatly limit their analytical commercial applications and require additional reagents for detection. In this work, the peptides that specifically bind with anti-benzothiostrobin monoclonal antibody (mAb) or benzothiostrobin-mAb immunocomplex were synthesized and conjugated with fluorescein isothiocyanate (FITC) as substitutes of the phage-displayed peptides to avoid their shortcomings and extend their applications. Competitive and noncompetitive fluorescence immunoassays (FIAs) for benzothiostrobin were developed by mAb coupling with magnetic nanoparticles as concentration elements and peptides conjugated with FITC as tracers. Compared with enzyme-linked immunosorbent assays, the FIAs reduced the number of steps from 6 to 2 and analysis time from more than 5 to 1.2 h. The competitive FIA showed the half-maximal inhibition concentration (IC50) of 16.8 ng mL-1 and detection range (IC10-IC90) of 1.0-759.9 ng mL-1, while the concentration of analyte producing 50% saturation of the signal (SC50) and detection range (SC10-SC90) of noncompetitive FIA were 93.4 and 5.9-788.2 ng mL-1, respectively. The average spiked recoveries were 68.33-98.50% and 73.33-96.67% for competitive and noncompetitive FIAs, respectively. The FIAs showed good correlation with high-performance liquid chromatography for the detection of benzothiostrobin in authentic samples. Graphical abstract Development of competitive and noncompetitive fluorescence immunoassays for benzothiostrobin by using monoclonal antibody coupling with magnetic nanoparticles as concentration elements and peptides conjugated with fluorescein isothiocyanate as tracers.

SUBMITTER: Chen H

PROVIDER: S-EPMC7263471 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides.

Chen He H Yang Qian Q Ding Yuan Y Vasylieva Natalia N Bever Candace S CS Hua Xiude X Wang Minghua M Hammock Bruce D BD

Analytical and bioanalytical chemistry 20181126 2

Phage-displayed peptides have been proven to be powerful reagents for competitive and noncompetitive immunoassays. However, they are unconventional reagents, which greatly limit their analytical commercial applications and require additional reagents for detection. In this work, the peptides that specifically bind with anti-benzothiostrobin monoclonal antibody (mAb) or benzothiostrobin-mAb immunocomplex were synthesized and conjugated with fluorescein isothiocyanate (FITC) as substitutes of the ...[more]

PMID: 30478514

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Project description:Fluorescence-based detection methods are being increasingly utilized in molecular analyses. Sequence-specific fluorescently-labeled probes are favored because they provide specific product identification. The most established fluorescence-based detection systems employ a resonance energy transfer mechanism effected through the interaction of two or more fluorophores or functional groups conjugated to oligonucleotide probes. The design, synthesis and purification of such multiple fluorophore-labeled probes can be technically challenging and expensive. By comparison, single fluorophore-labeled probes are easier to design and synthesize, and are straightforward to implement in molecular assays. We describe herein a novel fluorescent strategy for specific nucleic acid detection and genotyping. The format utilizes an internally quenched fluorescein-oligonucleotide conjugate that is subsequently dequenched following hybridization to the target with an attendant increase in fluorescence. Reversibility of the process with strand dissociation permits Tm-based assessment of bp complementarity and mismatches. Using this approach, we demonstrated specific detection, and discrimination of base substitutions of a variety of synthetic nucleic acid targets including Factor V Leiden and methylenetetrahydrofolate reductase. We further demonstrated compatibility of the novel chemistry with polymerase chain reaction by amplification and genotyping of the above listed loci and the human hemoglobin beta chain locus. In total, we analyzed 172 clinical samples, comprising wild-type, heterozygous and homozygous mutants of all three loci, with 100% accuracy as confirmed by DNA sequencing, established dual hybridization probe or high performance liquid chromatography-based methods. Our results indicate that the dequenching-based single fluorophore format is a feasible strategy for the specific detection of nucleic acids in solution, and that assays using this strategy can provide accurate genotyping results.

Dataset Information

Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides.

Publications

Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides.

Similar Datasets

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