<meta charset="utf8"></head><body>使用荧光成像和 EPR 检测血小板中的超氧阴离子

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH)</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-02.1-50mg</td>
			<td>Reagent for EPR (spin probe)</td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences&#160;</td>
			<td>NA</td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Merck/Sigma</td>
			<td>A7030</td>
			<td>For &#956;-slide coating</td>
		</tr>
		<tr>
			<td>Bruker E-scan M (Noxyscan)</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>&#160;NOX-E.11-BES&#160;</td>
			<td>EPR spectrometer</td>
		</tr>
		<tr>
			<td>Catalase&#8211;polyethylene glycol (PEG-Cat.)</td>
			<td>Merck/Sigma</td>
			<td>C4963</td>
			<td>Hydrogen peroxide scavenger (specificity control)</td>
		</tr>
		<tr>
			<td>ChronoLog Model 490+4</td>
			<td>Labmedics/Chronolog</td>
			<td>NA</td>
			<td>Aggregometer</td>
		</tr>
		<tr>
			<td>CM radical</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-20.1-100mg&#160;</td>
			<td>Reagent for EPR (calibration control)</td>
		</tr>
		<tr>
			<td>deferoxamine&#160;</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-09.1-100mg&#160;</td>
			<td>Reagent for EPR</td>
		</tr>
		<tr>
			<td>diethyldithiocarbamate (DETC)&#160;</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-10.1-1g&#160;</td>
			<td>Reagent for EPR</td>
		</tr>
		<tr>
			<td>Dihydroethidium</td>
			<td>Thermo Fisher Scientifics</td>
			<td>D11347</td>
			<td>Superoxide anion probe</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Merck/Sigma</td>
			<td>34869</td>
			<td>For stock solution preparation&#160;</td>
		</tr>
		<tr>
			<td>EPR sealing wax plates</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-A.3-VPM</td>
			<td>Consumable for EPR</td>
		</tr>
		<tr>
			<td>EPR-grade water</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-07.7.1-0.5L&#160;</td>
			<td>Reagent for EPR</td>
		</tr>
		<tr>
			<td>Fibrinogen from human plasma</td>
			<td>Merck/Sigma</td>
			<td>F4883</td>
			<td>For &#956;-slide coating</td>
		</tr>
		<tr>
			<td>FITC anti-human CD41 Antibody</td>
			<td>BioLegend</td>
			<td>303704</td>
			<td>Platelet-specific staining for flow cytometry</td>
		</tr>
		<tr>
			<td>Glass cuvettes&#160;</td>
			<td>Labmedics/Chronolog</td>
			<td>P/N 312</td>
			<td>Consumable for incubation in aggregometer</td>
		</tr>
		<tr>
			<td>Horm Collagen</td>
			<td>Labmedics/Chronolog</td>
			<td>P/N 385</td>
			<td>For platelet stimulation</td>
		</tr>
		<tr>
			<td>ImageJ&#160;</td>
			<td>National Institutes of Health (NIH)</td>
			<td>NA</td>
			<td>ImageJ 1.53t (Wayne Rasband)</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Merck/Sigma</td>
			<td>I7378</td>
			<td>For platelet isolation</td>
		</tr>
		<tr>
			<td>Micropipettes DURAN 50&#181;l</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-G.6.1-50&#181;L</td>
			<td>Consumable for EPR</td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrochloride</td>
			<td>Merck/Sigma</td>
			<td>P2658</td>
			<td>For &#956;-slide coating</td>
		</tr>
		<tr>
			<td>Prostaglandin E1 (PGE1)</td>
			<td>Merck/Sigma</td>
			<td>P5515</td>
			<td>For platelet isolation</td>
		</tr>
		<tr>
			<td>Sodium citrate (4% w/v solution)</td>
			<td>Merck/Sigma</td>
			<td>S5770</td>
			<td>For platelet isolation</td>
		</tr>
		<tr>
			<td>Stirring bars (Teflon-coated)</td>
			<td>Labmedics/Chronolog</td>
			<td>P/N 313</td>
			<td>Consumable for incubation in aggregometer</td>
		</tr>
		<tr>
			<td>Superoxide dismutase&#8211;polyethylene glycol (PEG-SOD)</td>
			<td>Merck/Sigma</td>
			<td>S9549</td>
			<td>Superoxide anion scavenger (specificity control)</td>
		</tr>
		<tr>
			<td>Thrombin from human plasma</td>
			<td>Merck/Sigma</td>
			<td>T6884</td>
			<td>For platelet stimulation and &#956;-slide coating</td>
		</tr>
		<tr>
			<td>VAS2870</td>
			<td>Enzo Life Science</td>
			<td>BML-EI395</td>
			<td>NOX inhibitor</td>
		</tr>
		<tr>
			<td>Zeiss 510 LSM confocal microscope</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>Confocal microscope</td>
		</tr>
	</tbody>
</table>

alternative methods for the detection of superoxide anion generation in platelets

Read this Scientific Article Protocol about Alternative Methods for the Detection of Superoxide Anion Generation in Platelets at JoVE.com

<meta charset="utf8"></head><body>作者聚焦：ROS 检测的创新技术及其对血小板研究的影响

检测血小板中超氧阴离子生成的替代方法 

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ...

alternative-methods-for-detection-superoxide-anion-generation

Research

JoVE Journal

Immunology and Infection

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

459 次观看.  South Mimms laboratories. 我们的研究范围是了解活性氧的产生如何调节血小板功能和调节止血反应。在我们的工作中，可靠地检测关键的活性氧物质——超氧阴离子至关重要。检测超氧阴离子的最常见技术是使用 DCFDA 作为荧光探针的流式细胞术。这些作为限制，它会诱导活性氧的释放，从而导致数据不可靠。它不允许区分不同的活性氧，也不允许量化血小板活性氧。?...

视频: 作者聚焦：ROS 检测的创新技术及其对血小板研究的影响

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

定量检测抗体诱导补体激活的方法对红血细胞

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

生物能量和氧化突发:协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

双色抗原微阵列产生的IgG和IgM自身抗体的同时检测

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...