Comparison of an ELISA and two reverse transcription polymerase chain reaction methods for norovirus detection

Kele, Beatrix; Lengyel, György; Deák, Judit

doi:10.1016/j.diagmicrobio.2011.04.002

Cited by 36 publications

(18 citation statements)

References 18 publications

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“…Our results showed that Nano-85 could detect the current pandemic GII.4 norovirus virions in clinical stool specimens from patients with sporadic gastroenteritis using a sandwich ELISA format, although the detection rate was low. On the other hand, most ELISA detection kits currently are used for screening outbreak specimens and have low detection rates with sporadic specimens (18,(39)(40)(41). Therefore, these results showed that Nano-85 might function as a valuable reagent in a diagnostic detection kit, although we acknowledge that additional ELISA formats will need to be tested in order to improve the detection rate.…”

Section: Discussionmentioning

confidence: 95%

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Koromyslova

Hansman

2015

J Virol

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Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ϳ10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability. IMPORTANCEWe characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich enzyme-linked immunosorbent assay. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 not only will work as a diagnostic reagent in norovirus detection systems but also could function as a broadly reactive GII norovirus antiviral.

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Section: Discussionmentioning

confidence: 95%

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Koromyslova

Hansman

2015

J Virol

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“…The lack of broad reactivity by antibodies to human NoV strains has been well documented (Burton-MacLeod et al, 2004;Shiota et al, 2007), and for this reason, enzyme immunoassays display poor sensitivity (Costantini et al, 2010;Kele et al, 2011). Other candidate NoV ligands have been explored, such as putative NoV infection co-factors known as histo-blood group antigens (HBGAs) (Cannon and Vinjé, 2008;Harrington et al, 2004) and porcine gastric mucin, which contains some HBGAs (Pan et al, 2012;Tian et al, 2008); peptides (Rogers et al, 2013); and single chain antibodies (Huang et al, 2014).…”

Section: Introductionmentioning

confidence: 98%

Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain

Moore

Escudero-Abarca

Suh

et al. 2015

Journal of Biotechnology

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Human noroviruses (NoV) are the leading cause of acute viral gastroenteritis worldwide. Significant antigenic diversity of NoV strains has limited the availability of broadly reactive ligands for design of detection assays. The purpose of this work was to produce and characterize single stranded (ss)DNA aptamers with binding specificity to human NoV using an easily produced NoV target-the P domain protein. Aptamer selection was done using SELEX (Systematic Evolution of Ligands by EXponential enrichment) directed against an Escherichia coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of 14 virus-like particles (VLPs) showed these aptamers had broad reactivity and exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs. Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested. Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P domain protein as a functional target for the selection of nucleic acid aptamers that specifically bind and broadly recognize diverse human NoV strains.

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“…Current approaches for detecting norovirus in clinical and environmental samples utilize a combination of electron microscopy techniques [20][21][22][23][24], molecular detection assays [7,10,17,[25][26][27] and immunological methods [25,[28][29][30][31][32][33]. Each approach strives for maximizing sensitivity and specificity while minimizing the time for detection [34].…”

Section: Introductionmentioning

confidence: 99%

“…ELISAs can analyze a myriad of pathological samples using multiple screening modes and reporter molecules, giving the assay broad versatility, but their need for high viral loads [5,27,30] limits the assays' application mostly in clinical settings. Despite their relatively lower sensitivity when compared to PCR-based methods [5,20], ELISA-based assays have been used to detect norovirus in human stool samples [5,25,[36][37][38][39][40][41][42][43], human sera [29], and food samples [26,31,32,44,45] Using norovirus GI.1 (Norwalk) virus-like particles (VLPs) as a model viral system, the objective of this study was to develop and evaluate several ELISA-based assays for rapid detection of varying concentrations of GI.1 VLPs (0.037-3.7 μg/mL). HuNoV VLPs are replication-incompetent, macromolecular protein assemblies with capsid structures and antigenic properties resembling those of innate norovirus particles [46][47][48].…”

Section: Introductionmentioning

confidence: 99%

Design and Evaluation of Three Immuno-based Assays for Rapid Detection of Human Norovirus Virus-like Particles

Broglie¹,

Moore²,

Jaykus³

et al. 2014

J Anal Bioanal Tech

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This study designed and evaluated three versatile immuno-based assays for the rapid detection of GI.1 norovirus virus-like particles at low (0-3.0 μg/mL) levels: 1) enzymatic absorbance-based ELISAs, 2) a fluorescentbased immunoassay, and 3) a "signal-down" capture ELISA. Variables including controllable variations in assay format (indirect or sandwich), assay time, binding sequence, and reporter molecule (fluorophore or enzyme) were thoroughly investigated and optimized in all three assays. Selectivity tests in the three-hour absorbance-based ELISA using VLPs representing two GI and two GII strains indicated the assays were selective to GI strains over GII strains. The three-hour enzymatic absorbance-based assay turned out to be a robust and rapid method capable of detecting GI.I VLPs in the range of 0.037 to 0.555 μg/mL, and the three-hour fluorescent immunoassay was capable of detecting VLPs in a high concentration range of 0.5-2.0 μg/mL under optimized conditions. The "signal-down" capture ELISA was conceptually demonstrated for the detection of VLPs at concentrations in excess of 1.0 μg/ mL, but did not appear to be suitable for quantifying VLPs under its current conditions. The methods reported here provide proof-of-concept that various ELISA-type approaches could be further developed to provide robust norovirus detection assays having various detection ranges, limits, and linearity.

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Comparison of an ELISA and two reverse transcription polymerase chain reaction methods for norovirus detection

Cited by 36 publications

References 18 publications

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain

Design and Evaluation of Three Immuno-based Assays for Rapid Detection of Human Norovirus Virus-like Particles

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