An Enzyme-Linked Aptamer Sorbent Assay to Evaluate Aptamer Binding

Moore, Matthew D.; Escudero-Abarca, Blanca; Jaykus, Lee‐Ann

doi:10.1007/978-1-4939-6857-2_18

Cited by 5 publications

(4 citation statements)

References 10 publications

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“…To show the utility of aptamer-based virus detection, we developed an enzyme linked chemiluminescence sandwich assay. , This is a proof-of-concept demonstration of the use of aptamers in the rapid and sensitive detection of DCV. Such an assay could be used for screening infected flies.…”

Section: Resultsmentioning

confidence: 99%

Application of the Open qPCR Instrument for the in Vitro Selection of DNA Aptamers against Epidermal Growth Factor Receptor and Drosophila C Virus

et al. 2018

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The low-cost Open qPCR instrument can be used for different tasks in the aptamer selection process: quantification of DNA, cycle course optimization, screening, and final binding characterization. We have selected aptamers against whole Drosophila C virus (DCV) particles and recombinant epidermal growth factor receptor (EGFR). We performed systematic evolution of ligands by exponential enrichment (SELEX) using the Open qPCR to optimize each amplification step. The Open qPCR instrument identified the best aptamer candidate. The Open qPCR has the capacity to perform melt curves, and we used this function to perform thermofluorimetric analysis (TFA) to quantify target-aptamer binding. We confirmed target-aptamer binding using flow cytometry. A sandwich type luminescence bioassay based on our anti-DCV aptamer was sensitive to DCV and did not respond to a related virus, demonstrating that our selected anti-DCV aptamer can be used to specifically detect DCV.

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Section: Resultsmentioning

confidence: 99%

Application of the Open qPCR Instrument for the in Vitro Selection of DNA Aptamers against Epidermal Growth Factor Receptor and Drosophila C Virus

et al. 2018

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“…An ELASA was used to determine relative binding affinity of each aptamer to both Norwalk and Tulane virus VPg proteins based on previously reported procedures [ 5 , 18 , 33 ], using glutathione coated 96-well plates pre-blocked with bovine serum albumin (BSA) and SuperBlock buffer (Thermo Scientific, Waltham, MA, USA). Crude protein extracts for GST-VPg (Norwalk virus), GST-VPg (Tulane virus), and GST-only were diluted to 1% solutions in PBS with 0.05% Tween 20 (PBST) prior to use for the binding assay.…”

Section: Methodsmentioning

confidence: 99%

Generation of Nucleic Acid Aptamer Candidates against a Novel Calicivirus Protein Target

Faircloth

Moore

Stoufer

et al. 2021

Viruses

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Human norovirus is the leading cause of foodborne illness globally. One of the challenges in detecting noroviruses is the identification of a completely broadly reactive ligand; however, all detection ligands generated to date target the viral capsid, the outermost of which is the most variable region of the genome. The VPg is a protein covalently linked to the viral genome that is necessary for replication but hitherto remains underexplored as a target for detection or therapeutics. The purpose of this work was to generate nucleic acid aptamers against human norovirus (Norwalk) and cultivable surrogate (Tulane) VPgs for future use in detection and therapeutics. Eight rounds of positive-SELEX and two rounds of counter-SELEX were performed. Five and eight unique aptamer sequences were identified for Norwalk and Tulane VPg, respectively, all of which were predicted to be stable (∆G < −5.0) and one of which occurred in both pools. All candidates displayed binding to both Tulane and Norwalk VPg (positive:negative > 5.0), and all but two of the candidates displayed very strong binding (positive:negative > 10.0), significantly higher than binding to the negative control protein (p < 0.05). Overall, this work reports a number of aptamer candidates found to be broadly reactive and specific for in vitro-expressed VPgs across genus that could be used for future application in detection or therapeutics. Future work characterizing binding of the aptamer candidates against native VPgs and in therapeutic applications is needed to further evaluate their application.

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“…Binding of aptamer AG3 to MNV particles was investigated according to a previously reported protocol. 15 Briefly, the wells containing MNV of 1, 10, 100, or 1,000 plaque-forming unit (PFU) mL ¹1 or PBS (negative control) were blocked with 200 µl of 5% skim milk in PBS-Tween 20 (0.05%, vol/vol; PBST) with a 10 nM mixture of unrelated PCR primers (Listeria monocytogenes primers hlyQF/R and L23SQF/R) 16 overnight at 4°C with agitation (250 rpm). The plates were washed thrice with 200 µL of PBST and then incubated with 100 µL of 1 µM biotinylated AG3 aptamer for 1 h with agitation.…”

Section: Enzyme-linked Aptamer Sorbent Assay (Elasa)mentioning

confidence: 99%

Improvement of Electrochemical Conditions for Detecting Redox Reaction of K<sub>3</sub>[Fe(CN)<sub>6</sub>] toward the Application in Norovirus Aptasensor

et al. 2020

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Electrochemical biosensors have attracted significant attention as a novel tool for the sensitive detection of pathogens and contaminants, with the potential capability of rapid, on-site diagnosis. In this study, we intended to improve the sensitivity of electrochemical biosensor using Fe(CN) 63−/4− as redox marker, toward its application to norovirus aptasensors. Although many researchers have developed electrochemical aptasensors for various analytes using redox markers, the reported electrochemical conditions under which an aptasensor was examined varied across multiple studies. Here, we performed square-wave voltammetry (SWV) for electrodes modified with aptamer specific to murine norovirus and compared the reduction peak currents of Fe(CN) 63− under various conditions. Effects of working electrode materials and NaCl and [Ru(NH 3 ) 6 ]Cl 3 concentration in the electrolyte were examined. Among conditions we tested, the best sensitivity was obtained using a screen-printed gold electrode in an electrolyte containing 1 M NaCl with 4 mM K 3 [Fe(CN) 6 ], in which the concentration of murine norovirus showed linearity with SWV peak current. This study provided useful information on the electrochemical measurement conditions regarding the development of electrochemical aptasensors.

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An Enzyme-Linked Aptamer Sorbent Assay to Evaluate Aptamer Binding

Cited by 5 publications

References 10 publications

Application of the Open qPCR Instrument for the in Vitro Selection of DNA Aptamers against Epidermal Growth Factor Receptor and Drosophila C Virus

Application of the Open qPCR Instrument for the in Vitro Selection of DNA Aptamers against Epidermal Growth Factor Receptor and Drosophila C Virus

Generation of Nucleic Acid Aptamer Candidates against a Novel Calicivirus Protein Target

Improvement of Electrochemical Conditions for Detecting Redox Reaction of K<sub>3</sub>[Fe(CN)<sub>6</sub>] toward the Application in Norovirus Aptasensor

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