我们要感谢Jean-François Papon，Bruno Louis，Estelle Escudier以及巴黎东部PCD诊断中心的所有团队成员在访问他们的PCD诊断中心期间的可用性和热烈的欢迎，以及众多的交流。我们还要感谢罗伯特·赫斯特和莱斯特PCD中心的所有团队成员的欢迎和时间，建议和专业知识。

这些作者没有什么可透露的。

本文旨在为使用鼻刷样本的CFA提供标准操作程序，并在COVID-19大流行期间根据适当的感染控制考虑进行调整。PCD诊断具有挑战性，根据国际建议，目前需要一组不同的诊断测试，包括鼻一氧化氮测量，使用DHSV的CFA，使用透射电子显微镜（TEM）的睫状超微结构分析，使用免疫荧光标记睫状蛋白，以及PCD致病基因4，37的基因检测。目前，没有单一的测试可?...

原发性纤毛运动障碍（PCD）是一种遗传性异质性活动性纤毛病，其中呼吸纤毛静止，缓慢或运动障碍，导致粘液纤毛清除受损和慢性耳窦肺疾病1，2，3，4。PCD的临床表现是婴儿早期开始的慢性湿咳和慢性鼻塞，复发性或慢性上呼吸道和下呼吸道感染导致支气管扩张，以及复发性或慢性中耳炎和鼻窦炎5，6，7。大约一半的PCD患者表现为器官偏侧性缺陷，例如位位逆位或位位模糊。一些患者还由于男性精子不动和女性输卵管不动纤毛而出现不孕问题1，2，8。PCD 很少见，但患病率难以定义，范围为 1：10，000 至 1：20，0009，10。然而，由于诊断困难和缺乏临床怀疑，PCD 的实际患病率被认为更高。PCD的症状与其他急性或慢性呼吸系统疾病的常见呼吸表现相似，确认诊断的诊断挑战是众所周知的，导致治疗和随访不足2，5，9，11。
使用数字高速视频显微镜（DHSV）的睫状视频显微镜是PCD4，8，12，13的诊断工具之一。DHSV被认为是进行睫状功能分析（CFA）的最佳方法，包括睫状搏动频率（CBF）和搏动模式（CBP）分析2，14，15，16。DHSV使用活的呼吸道上皮，通常通过鼻刷获得13。
鉴于当前的COVID-19爆发，PCD诊断的确认现在变得更加重要，因为有证据表明，潜在的呼吸道疾病可能导致COVID-19感染后更糟糕的结果17，18。与一般人群相比，在当前大流行期间安全有效的PCD诊断服务也将使确诊的PCD患者受益于额外的保护措施19。
COVID-19的传播主要通过飞沫传播20。鼻样本20中的高病毒载量表明无症状（或症状轻微）患者的传播可能性很高。此外，如果病毒颗粒被雾化，它们会在空气中停留至少 3 小时21。因此，呼吸系统医护人员在进行临床护理和诊断技术样本采集时会暴露在高病毒载量中22。此外，操纵活体呼吸道样本会使技术人员暴露于 COVID-19 污染中。虽然正在实施针对护理COVID-19患者的呼吸内科医生和耳鼻喉外科医生的最佳实践建议23，但缺乏在COVID-19大流行期间实施DHSV的建议。
为了继续提供PCD诊断服务，同时确保医护人员（进行样本采集）和技术人员（进行样本处理）的安全，必须在COVID-19大流行期间调整睫状视频显微镜方案。睫状视频显微镜技术目前仅限于研究服务和专业诊断中心，因为CFA需要广泛的培训和经验。此外，目前，使用DHSV4，13处理和分析样品缺乏标准化和精确的操作程序。
本文的目的是描述DHSV的标准操作程序，特别是感染控制措施和采样和处理活鼻上皮时的安全性。尽管目前COVID-19爆发，但这将使高质量的PCD诊断和护理得以继续。

<table>
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>FisherScientific</td>
			<td>352096</td>
			<td>15 ml High-Clarity Polypropylene Conical Tube with lid</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>LONZA</td>
			<td>17-836E</td>
			<td>Antifungal solution</td>
		</tr>
		<tr>
			<td>Blakesley-weil nasal forceps</td>
			<td>NOVO SURGICAL</td>
			<td>E7739-12</td>
			<td>Used to hold the brush to perform the nasal brushing</td>
		</tr>
		<tr>
			<td>Bronchial cytology brush</td>
			<td>CONMED</td>
			<td>129</td>
			<td>Used for nasal brushing</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>NUOVA APTACA</td>
			<td>2150/SG</td>
			<td>Used for COVID-19 testing</td>
		</tr>
		<tr>
			<td>Digitial high-speed videomicroscopy camera</td>
			<td>IDTeu Innovation in motion</td>
			<td>CrashCam Mini 1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>ThermoScientific</td>
			<td>12372098</td>
			<td>Microscope slides used to create the visualization chamber</td>
		</tr>
		<tr>
			<td>Heated Box</td>
			<td>IBIDI cells in focus</td>
			<td>10918</td>
			<td>Used to heat the sample</td>
		</tr>
		<tr>
			<td>Inverted Light microscope</td>
			<td>Zeiss</td>
			<td>AXIO Vert.A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Heater</td>
			<td>TOKAI HIT</td>
			<td>TPiE-LH</td>
			<td>Used to heat the oil immersion lens</td>
		</tr>
		<tr>
			<td>Medium 199 (M199), HEPES</td>
			<td>TermoFisher Scientific</td>
			<td>12340030</td>
			<td>Cell Culture Medium</td>
		</tr>
		<tr>
			<td>Motion Studio X64</td>
			<td>IDT Motion</td>
			<td>version 2.14.01</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Oil</td>
			<td>FischerScientific, Carl Zeiss</td>
			<td>11825153</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular cover slip</td>
			<td>VWR</td>
			<td>631-0145</td>
			<td>Used to cover the visualization chamber</td>
		</tr>
		<tr>
			<td>Spacer (Ispacer) 0.25 mm</td>
			<td>Sunjinlab</td>
			<td>IS203</td>
			<td>Used for the creation of the hermetic closed visualization chamber</td>
		</tr>
		<tr>
			<td>Square cover slip</td>
			<td>VWR</td>
			<td>631-0122</td>
			<td>Used for the creation of lab-built open visualization chamber</td>
		</tr>
		<tr>
			<td>Streptomycin/Penicillin</td>
			<td>FisherScientific, Gibco</td>
			<td>11548876</td>
			<td>Antiobiotics solution</td>
		</tr>
	</tbody>
</table>

nasal brushing sampling and processing using digital high speed ciliary videomicroscopy &ndash; adaptation for the covid-19 pandemic

原发性纤毛运动障碍（PCD）是一种遗传性活动性纤毛病，可导致严重的耳鼻肺疾病。由于不同诊断方式的挑战，PCD诊断经常被遗漏或延迟。睫状视频显微镜使用PCD诊断工具之一的数字高速视频显微镜（DHSV）被认为是进行睫状功能分析（CFA）的最佳方法，包括睫状搏动频率（CBF）和搏动模式（CBP）分析。然而，DHSV缺乏标准化的、公布的处理和分析样品的操作程序。它还使用活呼吸道上皮，这是 COVID-19 大流行期间的一个重要感染控制问题。为了在这场健康危机期间继续提供诊断服务，睫状视频显微镜检查方案已经过调整，以包括适当的感染控制措施。
在这里，我们描述了纤毛呼吸道样本采样和实验室处理的修订方案，重点介绍了为遵守COVID-19感染控制措施而进行的调整。描述了从16名健康受试者获得的鼻刷样品中CFA的代表性结果，根据该协议进行处理和分析。我们还说明了获得和处理最佳质量上皮纤毛条的重要性，因为不符合质量选择标准的样品现在确实允许CFA，这可能会降低该技术的诊断可靠性和效率。

为了说明该技术的效率，我们在一系列16名健康成年志愿者（5名男性，年龄范围22-54岁）中展示了CFA的结果。
在总共16名志愿者中，来自14名（4名男性，年龄范围24-54岁）的鼻刷样本提供了足够合适的上皮边缘，满足进行CFA所需的选择标准。从这14个鼻刷样本中，共记录了242个纤毛边缘，212个边缘符合定义的纳入标准并进行了分析。所有这些边缘都记录在侧向剖面中（记录并分...

Watch this Scientific Journal Video about Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy Ã¢â‚¬â€œ Adaptation for the COVID-19 Pandemic at JoVE.com

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

为了保证PCD诊断的成功和高质量的睫状功能分析，一种精确而仔细的呼吸上皮取样和处理方法至关重要。为了在COVID-19大流行期间继续提供PCD诊断服务，睫状视频显微镜检查方案已更新，以包括适当的感染控制措施。

使用数字高速睫状视频显微镜进行鼻刷采样和处理 – 适应 COVID-19 大流行

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

nasal-brushing-sampling-processing-using-digital-high-speed-ciliary

Research

JoVE Journal

Immunology and Infection

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

4.5K 次观看.  University of Liège. 为了保证PCD诊断的成功和高质量的睫状功能分析，一种精确而仔细的呼吸上皮取样和处理方法至关重要。为了在COVID-19大流行期间继续提供PCD诊断服务，睫状视频显微镜检查方案已更新，以包括适当的感染控制措施。

视频: Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy – Adaptation for the COVID-19 Pandemic

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

手指棒运动诱导淋巴细胞凋亡的测定血液抽样方法

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

科研

免疫与感染

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Granulocyte-dependent Autoantibody-induced Skin Blistering

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky's postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA 1. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies 2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA 1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.

In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.

粒细胞相关的自身抗体引起的皮肤起泡

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

可视化的非溶胞胞吐作用<em&gt;新型隐球菌</em&gt;从巨噬细胞用数码光学显微镜

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

侵染微生物利用数字条码平台的基因表达谱

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

人胰岛自身抗体的电化学发光测定

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...