Nanopeptamers for the Development of Small-Analyte Lateral Flow Tests with a Positive Readout

Vanrell, Lucía; González-Techera, Andrés; Hammock, Bruce D.; González‐Sapienza, Gualberto

doi:10.1021/ac3031114

Cited by 28 publications

(33 citation statements)

References 21 publications

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“…The midpoint of the titration curve, corresponding to the concentration of analyte giving 50% of signal saturation (SC 50 ) was 2.2 ± 0.3, and the limit of detection (LOD = analyte concentration giving a 10% increase over the zero signal) was 0.48 ng/mL. These parameters are essentially the same that were obtained with the nanopeptamers prepared with streptavidin-HRP functionalized with biotinylated peptides, 11 showing that the variations in the attachment point to the streptavidin oligomer (Figure 1 ) do not have a major effect in the overall avidity of the complex for the IC. When these values are compared to the performance of the competitive assay set up with the same antibody, SC 50 = 28 ± 1.1 ng/mL and LOD = 4.0 ng/mL, it becomes evident that the recombinant nanopeptamer assay performs with increased sensitivity, representing an improvement of about 13 and 8 fold, respectively.…”

Section: Resultssupporting

confidence: 61%

“…To work-around these limitations, we recently demonstrated that the phage particles can be substituted by commercial conjugates of streptavidin or avidin loaded with synthetic anti-IC peptides that contain a biotinylated lysine in their N-terminus. 11 These complexes, that we termed nanopeptamers, could be used to develop two-site noncompetitive assays for small molecules, which performed with similar sensitivity and specificity than their parent anti-IC phage particles.…”

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confidence: 99%

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Recombinant Streptavidin Nanopeptamer Anti-Immunocomplex Assay for Noncompetitive Detection of Small Analytes

et al. 2014

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Short peptide loops selected from phage libraries can specifically recognize the formation of hapten-antibody immunocomplexes and can thus be used to develop phage anti-immunocomplex assays (PHAIA) for noncompetitive detection of small molecules. In this study, we generated recombinant chimeras by fusing anti-immunocomplex peptides selected from phage libraries to the N- or C-termini of core streptavidin and used them to setup phage-free noncompetitive assays for the herbicide clomazone (MW 240 Da). The best conditions for refolding were optimized by a high throughput screening allowing to obtain tens of mg of purified protein per liter of culture. The noncompetitive assay developed with these chimeras performed with a 50% saturating concentration (SC50) of 2.2 ± 0.3 ng/mL and limit of detection (LOD) of 0.48 ng/mL. Values that are 13- and 8-fold better that those obtained for the SC50 and LOD of the competitive assay setup with the same antibody. Apart from the first demonstration that recombinant peptide-streptavidin chimeras can be used for sensitive immunodetection of small molecules with a positive readout, this new assay component is a highly standardized reagent with a defined stoichiometry, which can be used in combination with the broad option of existing biotinylated reagents offering a great versatility for the development of conventional immunoassay and biosensors. The utility of the test was demonstrated analyzing the clomazone runoff during the rice growing season in northern Uruguay.

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Section: Resultssupporting

confidence: 61%

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confidence: 99%

Recombinant Streptavidin Nanopeptamer Anti-Immunocomplex Assay for Noncompetitive Detection of Small Analytes

et al. 2014

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“…9 This indicates that the spatial distribution of the peptides in the VTX nanopeptamer allows attainment of a similar avidity interaction with the immobilized IC than that achieved with the streptavidin or the phage display. Moreover, using the midpoint of the assay titration curve for comparison, the SC 50 of the pA-VTX molinate assay was ∼13-fold more sensitive than the IC 50 (analyte concentration causing 50% inhibition) of the competitive ELISA set-up with the same antibody (IC50 69 ± 0.5 ng/mL).…”

Section: Results and Discusionmentioning

confidence: 91%

Shiga-Like Toxin B Subunit of Escherichia coli as Scaffold for High-Avidity Display of Anti-immunocomplex Peptides

Lassabe¹,

Rossotti²,

González-Techera³

et al. 2014

Anal. Chem.

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Small compounds cannot bind simultaneously to two antibodies, and thus, their immunodetection is limited to competitive formats in which the analyte is indirectly quantitated by measuring the unoccupied antibody binding sites using a competing reporter. This limitation can be circumvented by using phage-borne peptides selected for their ability to specifically react with the analyte–antibody immunocomplex, which allows the detection of these small molecules in a noncompetitive format (PHAIA) with increased sensitivity and a positive readout. In an effort to find substitutes for the phage particles in PHAIA, we explore the use of the B subunit of the Shiga-like toxin of Escherichia coli, also known as verotoxin (VTX), as a scaffold for multivalent display of anti-immunocomplex peptides. Using the herbicides molinate and clomazone as model compounds, we built peptide–VTX recombinant chimeras that were produced in the periplasmic space of E. coli as soluble pentamers, as confirmed by multiangle light scattering analysis. These multivalent constructs, which we termed nanopeptamers, were conjugated to a tracer enzyme and used to detect the herbicide–antibody complex in an ELISA format. The VTX–nanopeptamer assays performed with over a 10-fold increased sensitivity and excellent recovery from spiked surface and mineral water samples. The carbon black-labeled peptide–VTX nanopeptamers showed great potential for the development of a lateral-flow test for small molecules with a visual positive readout that allowed the detection of up to 2.5 ng/mL of clomazone.

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“…Therefore, various immunoassays, including enzyme-linked immunosorbent assay (Wei et al, 2018 ; Liu et al, 2019 ), lateral-flow immunochromatography (LFIC) (Li et al, 2010 ), and other novel immune sensors (Fernández et al, 2010 ; Zeng et al, 2019 ) have been developed for the detection of various antibiotic residues in milk. Of these assays, LFIC is the simplest method and the most rapid to carry out (Vanrell et al, 2013 ). Most LFIC experiments employ colloidal gold (CG) as labels to achieve qualitative or quantitative analysis of analytes (CháferPericás et al, 2010 ).…”

Section: Introductionmentioning

confidence: 99%

Functional Up-Conversion Nanoparticle-Based Immunochromatography Assay for Simultaneous and Sensitive Detection of Residues of Four Tetracycline Antibiotics in Milk

Chen

et al. 2020

Front. Chem.

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An ultrahigh-sensitivity lateral flow immunochromatography (LFIC) assay based on up-converting nanoparticles (UCNPs) was developed to carry out a multi-residue detection of tetracycline in milk. The sensitivity of the immunoassay was greatly improved by the use of a broad-spectrum monoclonal antibody attached to UCNPs to form a signal probe. Under the optimal conditions, the UCNP-LFIC assay enabled sensitive detection of tetracycline (TC) as well as of oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX) within 10 min, with IC 50 values of 0.32, 0.32, 0.26, 0.22 ng/mL, respectively. There was no cross-reactivity with ten other antibiotics. Similarly, we evaluated the experimental results for matrix effects. Experiments involving spiking showed the four tetracycline antibiotics displaying mean recoveries ranging from 93.95 to 111.90% with relative standard deviations (RSDs) of < 9.95%. The detection results of actual samples using the developed method showed a good correlation ( R 2 ≥ 0.98) with the results using high-performance liquid chromatography (HPLC). Thus, the assay can achieve an ultrahighly sensitive detection of antibiotics in milk, and can hence promote human health and provides promising applications in the bio-detection field.

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Nanopeptamers for the Development of Small-Analyte Lateral Flow Tests with a Positive Readout

Cited by 28 publications

References 21 publications

Recombinant Streptavidin Nanopeptamer Anti-Immunocomplex Assay for Noncompetitive Detection of Small Analytes

Recombinant Streptavidin Nanopeptamer Anti-Immunocomplex Assay for Noncompetitive Detection of Small Analytes

Shiga-Like Toxin B Subunit of Escherichia coli as Scaffold for High-Avidity Display of Anti-immunocomplex Peptides

Functional Up-Conversion Nanoparticle-Based Immunochromatography Assay for Simultaneous and Sensitive Detection of Residues of Four Tetracycline Antibiotics in Milk

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