We thank the Clinical Microbiology Service staff of the Memorial Sloan-Kettering Cancer Center for help in collecting clinical specimens. This study was supported in part by a research agreement between MSKCC and Alere Scarborough (SK2013-0262).

道德守则:使用遗留临床标本被批准，并遵循纪念斯隆 - 凯特琳癌症中心伦理审查委员会的指导方针。 1.在运行分析注:甲型和乙型流感检测已被批准用于鼻咽拭子标本和存储在病毒的传输介质鼻咽拭子。拭子被包括在试剂盒中，并应被用于最佳性能。然而，人造丝，泡沫，植绒拭子或聚酯鼻拭子也可用于收集鼻拭子样品。 <ol><li>收集鼻拭子样本，将拭子插入具有最明显的排水或者说是最拥挤的鼻孔。轻轻旋转，推拭子插入鼻孔，直至遇到阻力，并从鼻孔慢慢取出之前将针对鼻壁几次。存放拭子和装有3毫升病毒的传输介质（VTM）的小瓶运输他们。 注意:仔细注意appropr莱特标本采集必须被视为标本采集不足可能会产生错误的结果。 注意:拭子标本采集后尽早进行测试。然而，它们可在室温下保存长达2小时或在2-8℃下进行长达24小时的冷冻如果测试不是立即可用。新鲜采集的标本是理想的最佳测试性能。不当的样品处理，储存和/或运输可能产生错误的结果。 注:作为预防措施，执行此测试人员应该把所有的标本都当成潜在通过处理标本时注意以下事项普遍预防感染。 </li><li>将所有样品至室温测试之前。 注:所有的测试部件仅用于单次使用并且不应当被用来执行多个测试。不要混用不同批号试剂盒成分。不要使用试剂盒过去的到期日期。 </li><li>把蓝色样品接收器ROO测试前m温度。橙色测试碱可以无需加热到室温进行试验。 </li><li>留在箔包裹所有试验片直到即将使用前。通过按下仪器侧面的电源键打开仪器。输入用户ID，然后按"确定"。 </li></ol> 2.运行测试<ol><li>开始测试过程中，触摸"运行测试"在仪器屏幕上。通过屏幕上的键盘或条形码扫描仪，然后按Enter病人ID"确定"。打开盖子，然后插入橙色试验基地到橙色试验基地持有者。 </li><li>确认正确的测试显示屏幕，按下"确定"。将蓝色样品接收器插入蓝色样品接收机持有者。 注:在仪器放置，因为这将防止到达正确的工作温度下的洗脱缓冲液并可能影响考试成绩之前，不要打开示例接收器。 </li&gt; <li>等待样品接收器预热。当仪器提示，去掉铝箔封口，然后将病人拭子被测试到样品接收器。 注:当删除箔，将两个手指放在样品接收器的边缘，以确保它留在地方。 </li><li>大力混在液体中的棉签，持续10秒，按压棉签头靠在一侧的样品接收器，你混吧，帮助逐出拭子标本。按"确定"继续。如果存储在病毒的传输介质测试拭子，涡VTM，持续10秒，然后加入200μl的样品接收器。 </li><li>按白转移盒插入蓝色的样品接收器。继续按在样品接收，直到橙色指示灯上升。 </li><li>抬起，然后转送盒连接到测试基地。观察橙色指示灯下降，一旦盒传输连接正确。盖上盖子，不开，直到"测试完成"屏幕上显示的消息。 </li></ol> 3.质量控制<ol><li>触摸"运行QC测试"在主屏幕上。选择QC测试中运行。确认测试类型通过触摸"OK"，然后按照屏幕上的提示完成测试，用于测试的QC样品不一致。 </li></ol> 4.结果解释<ol><li>试运行结束后，观察与解释流感A，B，未知亚型的出现在屏幕上显示的测试结果。对于无效结果重复测试。 </li></ol> 5.保养和清洁<ol><li>清洁仪器和周围长凳用70％乙醇或10％的漂白剂溶液每日区域。喷在湿润的无绒布擦拭清洗液。不要在仪器上直接倒入解决方案。 </li></ol>

<ol>
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The authors have nothing to disclose.

流感病毒是发病率和死亡率的显著世界范围的原因。流感的快速和准确的诊断是主要键期间呼吸季节管理流感爆发中的一个。其他基于抗原的测试是快速和容易执行;然而，它们具有低的灵敏度13。另一方面，传统的分子测试具有改善的敏感性，但需要更多的有经验的化验师来执行，而且更昂贵。在这项研究中和协议中描述的流感A和B测定是流感A和B的CLIA-放弃，快速的分子测试的技术是基于等温扩增流感病毒A和B的检测和分化我们表明代表性的结果证明了流感A和B测定的性能存档，冷冻NPS VTM样本的面板上进行评价相比另一个基于分子的呼吸面板测定为。 （C T）值的标本，表明由该测定的检出率较低，用较低的病毒滴度相关联。该法有超过流感病毒具有90.2％的敏感性和98.1％的特异性的检测对乙型流感病毒的检测改进性能。这里的结果与先前公布的文献26，27，28是一致的。 这项研究有两个重要的限制。首先，将样品储存在VTM的平台上测试。然而，在样品进行测试时，平台没有FDA批准用于该样品类型。因此，稀释成VTM，有可能降低检出低滴度VTM样本。第二，用对流感A测试和B测定的标本已储存在-70℃下进行6至9个月他们被接收之后立即新鲜标本做测试前，而反相的测试。这是可能的冻结标本进一步降低流感检测样品英寸流感A＆B试剂盒几步简单执行。不过，也有必须进行适当的完成两个关键步骤，如果他们出现错误的用户可以排除故障。首先，当最初设置的测定中，转送暗盒必须被连接到测试基底，直到它们卡入到位。如果这两个件没有适当地固定，运行将失败，并且将需要被从开始重复。二，仪器需要预热2分钟添加样品之前。在此之后的预热时间，用户可以在30秒内加样，连接调用Cartridge的试验基地，并盖上盖子。如果时间经过之前这个步骤完成之后，该仪器将失败的运行和设置示例将必须从开始重复。 测定的局限性包括略微增加操作时间和较低的灵敏度相比，呼吸面板，并不亚型甲型流感另外，可以与测定中使用的唯一的样本类型是鼻咽拭子或存储在NPS病毒运送介质;测定为任何其他样品类型，例如下呼吸道标本的性能，并没有进行评估。要注意的是所有的在这个协议的步骤是关键的是重要的。从书面协议的任何修改或偏差可能会导致较低的性能或该测定的，并且必须由用户进行评估。该技术的优点是，该仪器具有占地面积小，只需要15分钟来执行，并且即使该技术基于分子的方法，不需要特殊的培训或资格来执行。在这项研究中评估了流感A和B测定具有作为替代RIDTs流感的诊断的潜力。该系统具有一个短总测试时间的优点，具有占地面积小，并且易于使用。总体而言，该仪器的设计适合于床边血糖检测。

流感病毒感染导致发病率和死亡率，每年1，2，3的显著量。单纯性流感的特点是宪法和呼吸道症状，如发热，肌痛，头痛和非生产性咳嗽4,5。老年人，幼儿，免疫功能低下患者，和患者的潜在合并症处于严重的并发症，如肺炎，心肌炎，中枢神经系统疾病，甚至导致死亡6,7风险较高。 流感感染是抗病毒治疗的症状出现后48小时内及时服用能减少疾病的严重程度和长度从8其他呼吸道病毒唯一的。流感的快速鉴定也已示出减少使用的不必要的抗生素9,10。另外，住院患者感染流感病毒必须放置在适当的感染控制预防措施隔离室。然而，所造成的非流感病毒呼吸道疾病可能难以从流感临床区分。出于这个原因，对于流感快速准确的诊断测试是用于临床患者管理非常重要。 数测定法可用于检测和流感病毒鉴定。快速流感抗原检测试验（RIDTs）被广泛应用于临床实践中作为点护理测试，因为它们易于使用，并提供在15至30分钟11，12的结果;然而，他们的敏感性有很大的不同取决于制造商和人口（10-80％）进行测试，并且流感类型和subtyPE 13，14，15。直接荧光测定（的DFA）提供超过RIDTs优异灵敏度，但处理时间大于（〜3小时），并且必须由熟练的技术人员16，17来完成。病毒培养一直是流感的诊断的金标准，并已改善了两个RIDTs和DFA的18灵敏度。然而，流感病毒培养可以从2-14ð任何地方来完成，减少在帮助病人管理19其效用。最后，核酸扩增试验（NAAT）已经取代培养技术在流感诊断新的金标准。 NAAT被认为具有在几个小时检测流感的最大灵敏度。然而，NAAT是最昂贵的测定和需要专门的设备和技术来执行5 <s了&gt;，20，21，22，23，24，25。 此处所描述的流感A和B的测定法是第一CLIA-放弃分子快速流感测试是容易获得的。此法的工作原理是采用使用特定流感引物，其次是目标检测与分子信标探针等温扩增核酸内切酶泥炭截短扩增反应（NEAR）。该测定区分流感A选自B，需要2分钟来设置和处理一个样品，并且总共需要15分钟来完成。 这里，我们目前的流感A＆B试验的协议。此外，我们还提供了一个样本数据集比较流感A对已归档的鼻咽拭子（NPS）的性能和B检测标本保存在病毒的传输介质（VTM）到其他呼吸道病原体面板检测。

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rapid molecular detection and differentiation of influenza viruses a and b

流感是在人中由流感病毒引起A和B的传染性呼吸系统疾病，并导致每年发病率和死亡率的一个显著量。的甲型和乙型流感检测是第一个可用的CLIA-放弃分子快速流感测试。流感A和B的测试工作通过采用等温扩增与流感特异性引物，接着目标检测的分子信标探针。这里，A型流感的性能和B测定冷冻，归档鼻咽拭子（NPS）试样储存在病毒运输介质（VTM）进行比较，以呼吸面板测定。 流感A和B测定的性能通过的结果相比较，以呼吸面板参考方法进行评价。总甲型流感病毒的敏感性为67.5％（95％CI（CI），56.6-78.5），特异性为86.9％（CI，71.0-100）。对于乙型流感病毒的检测，其敏感性和特异性分别为90.2％（CI，68.5-100）和98.8％（CI，68.5-100）表示。 该系统具有的显著缩短测试时间比其他任何现有的分子检测及简单，无吸管程序运行的优势一个完全集成的，封闭的，小尺寸的系统。总体而言，流行性感冒A和B测定本研究评估具有作为一个点 - - 关心快速流感诊断测试的可能性。

在这项研究中，归档NPS标本采自2013年NPS标本在3毫升住院患者提交2012年12月15日和3月1日之间的流感爆发期间与纪念斯隆 - 凯特琳癌症中心（MSKCC）流感样症状的收集VTM，并与检测呼吸道病毒（RP），其中包括流行性感冒A，A-1，A-3，和B小组在研究期间的分子检测常规的临床实践的一部分进行测试，3675 ​​NPS标本提交到MSKCC临床实验室进行检测。这些标本，45，425，37，77的药检呈阳性的流感病毒A-1，A-3，A-UN-subtypable（Au）和B，分别与RP。共有360阳性标本，从每个流感亚型的40个样品，37金。此外，1-2即到达实验室紧接之前或之后的每个阳性的样本阴性样品被选择用于后续TESTI与甲型和乙型流感检测纳克。 总体而言，甲型和乙型法检测79流感病毒A，37乙型流感病毒，240负，和4无效结果（内部控制失效）（ 表1）。共有46样本44只RP和两个正对甲型和仅有B试验阳性不一致的结果。在其上检测到33/43的44个不和谐的标本进行第三次基于分子流感检测（8流感病毒A，21金，2乙型流感病毒）呈阳性的RP和0/1阳性（流感病毒A）由甲型和乙型流感检测。两个或两个以上检测之间的协议被认为是一种准确的鉴定。 总甲型流感病毒的总体甲型和乙型流感检测灵敏度为67.5％（95％CI（CI），56.6-78.5）。对于甲型流感亚型的敏感性为A-1，84.6％（CI，58.5-100），A-<html> 3，90.2％（CI，62.8-100）和Au，21.9％（CI，19.6-24.1）。对于流感病毒A的总特异性为86.9％（CI，71.0-100）。流感病毒B测试的灵敏度和特异性分别为90.2％（CI，68.5-100）和98.8％（CI，68.5-100）。 总体而言，在与结果不一致试样的平均CT值比为一致的结果（16.4±0.48对25.7±0.51; P&gt; 0.000）获得的那些显著升高（ 图1）。 <img alt="图1" src="/files/ftp_upload/54312/54312fig1.jpg" /> 图1:RP法检测呈阳性和阴性，分别测定每个流感亚型样品测得的平均CT值。误差棒代表标准偏差。 **，P &lt;0.01; ***，P &lt;0.001。1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/54312/54312table1.jpg" /> 表1:流感A＆B在VTM的性能标本相比，参考结果。

Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

我们描述了一个快速，分子为基础的甲型和乙型流感检测。流感检测采用等温扩增与流感特异性引物，其次是目标检测与分子信标探针检测15分钟内每一个目标。的甲型和乙型流感检测是用户友好的，并要求最少的动手时间来执行。

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

rapid-molecular-detection-differentiation-influenza-viruses

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.5K 次观看.  Memorial Sloan Kettering Cancer Center. 我们描述了一个快速，分子为基础的甲型和乙型流感检测。流感检测采用等温扩增与流感特异性引物，其次是目标检测与分子信标探针检测15分钟内每一个目标。的甲型和乙型流感检测是用户友好的，并要求最少的动手时间来执行。 

视频: Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

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

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX比色法检测为流感的DNA芯片

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

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 ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...