The authors thank Dr. M. Matrosovich for providing the parent MDCK and MDCK-SIAT1 cell lines and Roche Pharmaceuticals for supplying the oseltamivir carboxylate.We also appreciate Dr. Anne Weston for valuable suggestions on the manuscript. This study was dependent upon the valued collaboration of the WHO National Influenza Centres and the WHO CCs within the WHO GISRS, who provided the influenza viruses used. This work was funded by the Medical Research Council through Programme U117512723.

注:以下协议的生物安全水平（BSL）是季节性流感病毒和BSL 3+为潜在的流感大流行的病毒BSL 2。病毒滴定法需要事先中和实验来决定病毒控制（VC）的病毒浓度。 1.病毒滴度注意:根据病毒的数量和重复的次数，一孔板可以设置具有以下灵活性:（1）病毒稀释可以沿任一的行或列（ 图1）被布置。每个病毒应该占据一个独立的行/列。 （2）病毒稀释增量是柔性的，但它应与在左上角的最高病毒浓度开始。 （3）上有重复的数量没有限制。 注:的Madin Darby狗肾细胞（MDCK）和MDCK-SIAT1（SIAT）细胞好心由M. Matrosovich博士，马尔堡，提供芨芨草许多10。盟细胞是与人的CMP-N-乙酰神经氨酸稳定转染的MDCK细胞:唾液酸（SA）的增强表达β半乳糖苷α-2,6-唾液酸转移酶基因α2-6Gal终止寡糖。 <ol><li>等分部分足以MDCK细胞或MDCK-盟细胞8到96孔板（200μl/孔）。孵育细胞在37℃，5％CO 2的2或3天以达到融合。传播补充有10％热灭活胎牛血清（FCS）和抗生素（100U / ml青霉素和100μg/ ml链霉素）的DMEM细胞。孵育盟细胞在37℃，5％的CO 2和1mg / ml G418的硫酸盐。 注:稀释倍数取决于经验和所使用的细胞系。细胞单层必须汇合（即没有细胞壁或MDCK细胞和MDCK-SIAT1细胞紧密堆积布局可见轮廓）在病毒接种时。稀释液的例子基于MDCK或MDCK-SIAT1细胞汇合烧瓶的传代培养于表1中给出。 </li><li>清洗细胞3次，用200微升每孔病毒生长培养基（VGM）的。消毒用70％乙醇的多层洗板30分钟，然后冲洗在洗涤前的无菌磷酸盐缓冲盐水（PBS）或VGM。 </li><li>吸出VGM，立即加入50微升VGM的每个孔中。 </li><li>添加900微升VGM到每个6无菌试管中（或更少，这取决于测试的病毒和重复数）。吸取100微升病毒进入第一管，拌匀，并连续稀释，改变每个稀释度之间的提示。重复每个病毒滴定。 注意:这将给予10 -1病毒稀释至10-6。 </li><li>添加每种病毒稀释液50微升，起始于最高稀释度（1×10 -5 图1），在9重复的孔中（列9和10中的图1）6孔板中。 </li><li>放置在37℃的板2-3小时以使病毒感染的细胞。 注:A 2至3小时培养是必要的，以确保在接种病毒有足够的时间来达到单层。 </li><li>准备覆盖（10毫升/板） 注意:覆盖由5毫升的2×DMEM，胰蛋白酶（2微克/毫升的最终浓度），20微升的1毫克/毫升的库存，和5ml纤维素棉短绒（见材料清单）的。 </li><li>取出接种。 </li><li>加覆盖（200微升至每孔）。在37℃孵育过夜的，不受干扰。 注:病毒出芽发生之后大约4小时，因此重要的是，该板未在此时间后的干扰。 </li><li>吸从井中覆盖。 </li><li>加入冰冷的4％多聚甲醛的PBS（200微升/孔）。将它们放置在4℃下30分钟，或在室温下搅拌20分钟。 注:PBS A是自然pH值的磷酸盐缓冲盐水，10去˚F氯化钠0.25g的氯化钾，1.437克的 Na 2 HPO 4的0.25g的KH 2 PO 4，1升蒸馏水（见材料清单）。 注意:吸入和吞食可能造成灼伤时，多聚甲醛是有害的。还有具有致癌作用的证据有限，和皮肤接触后可能会引起过敏。 </li><li>吸出多聚甲醛，并用PBS洗涤板两次（200μl/孔）。 </li><li>存储在PBS A中的板在4℃下以供将来使用，或与下列步骤进行。 </li><li>添加透化缓冲液（100μl/孔）。离开它在室温下30分钟。 </li><li>用PBS洗两次板（200微升/孔）。 </li><li>加入50微升一抗，抗流感A型鼠标单克隆抗体（1:1,000 ELISA缓冲液），每口井。孵育它在室温下1小时（或4℃过夜），在摇动。 </li><li>在100μl洗涤缓冲液（0.05％吐温80在PBS中A，V / V）的p洗3次呃好。孵育它在室温下洗涤之间5分钟。另外，洗3次，而无需使用每孔300微升孵化。 </li><li>加入50μl的第二抗体的山羊抗小鼠IgG（H + L）HRP缀合物（1:1000在ELISA缓冲液），每孔。孵育它在室温下搅拌1小时，用摇动。 </li><li>如在步骤1.17中所述洗3次。 </li><li>添加底物（50μl/孔）。孵育，在室温下30分钟或直至蓝色的发展是清晰可见。 </li><li>停止反应，用200μl每孔蒸馏水洗涤平板两次，在室温下温育的洗涤之间2 -3分钟。 </li><li>空气干燥板，并将其存储在一个黑暗的地方（ 例如，包装在铝箔）。 </li></ol> 2.定量滴定<ol><li>放置在平板扫描仪的扫描区一个孔板， 如图2a所示。使用L形的位置限制确保最佳的和可重复的成像位置。 注意:可以将图像两个著名板在一次扫描。 </li><li>扫描图像。 注:设置示于表2中 。 </li><li>运行"多孔板阅读器"的软件来计算所需的病毒浓度（ 图3）。 <ol><li>点击"加载图像"按钮，加载图像。滑入"全球阈值"选项卡以调整采样阈值直方图的红条。点击"更新"按钮来检查图像上的效果。 </li><li>勾选"计算中和/滴定"框。点击"采集"按钮以量化的ICP。点击"保存"按钮，在出现提示时保存采样结果。 注:在采样过程后，如果"计算中和/滴定"框中打勾自动出现一个名为"中和滴定与计算"新窗口。 </li><li>负载或输入井板定义地图。表明在"滴定:最优病毒种群（％）"的阈值（ 例如，30％）。选择"滴定进程"选项卡来计算滴定结果。检查滴定结果。点击"保存并关闭"按钮，保存滴定结果。 注:请参阅补充S1有关软件的详细说明。可根据要求定制的软件的副本。 注:通常，最好的病毒稀释度为50％左右（20-85％）细胞总数的ICP斑块还原法产量每孔11之内。 </li></ol></li></ol> 3.病毒中和注:计算的中和测定所需板的数量。每个板可容纳一个病毒和一些抗血清。 注意:根据抗血清的数量和重复的次数，一孔板可以设置与第Ë以下灵活性:（1）血清稀释度可以沿任一行或一列（ 图4）被布置。每个血清应该占据一个独立的行或列。 （2）血清稀释度的增量是柔性的，但它应与在板的左上角的最高血清浓度开始。 （3）重复的数量平衡的抗血清的数量。更多的重复可以帮助理顺实验的变化。 （4）在VC的数量和单元控制（CC）的重复是柔性的，但它们也应该是在单独的行或列的数目。据预计，VC副本的数目比抗血清的显著更大。 <ol><li>制备细胞单层，如在步骤1.1-1.3，2或3天提前。 </li><li>加入50μlVGM的至板的各孔中。 </li><li>分别用11和12列的VC和CC。添加1:20受体破坏酶（RDE）处理血清的50微升等分试样到科拉姆的第一行（A）的NS 1-10。 注:对于每种病毒和血清结合，该试验一式两份进行，从而一个板可，例如，包含一种病毒对五种抗血清进行测试。 </li><li>执行从A行从行H.转移50微升到行H（列1-10 图4），并丢弃50微升2倍系列稀释</li><li>加入50μl稀释液向CC柱（ 图4中第12列）的各孔中。 </li><li>加入50μl病毒至板的各孔中，除了用于CC柱（列1-11，行AH中图4）。 </li><li>孵育它在37℃下2-3小时。取出接种。加覆盖（200微升）到每个孔中。过夜孵育它在37℃下，原状。修复和染色板作为病毒滴度（步骤1.11-1.22）。 </li></ol> 4.中和定量<ol><li>放置一个孔板在平板扫描仪的扫描区，如图Figu重新2A。使用L形位置的限制，以保证最佳的和可重复的成像位置。 注意:可以将图像两个著名板在一次扫描。 </li><li>扫描板，如在步骤2.2中描述。 </li><li>运行"多孔板阅读器"的软件来计算所需的病毒滴度（ 图3，步骤2.3）。 <ol><li>点击"加载图像"按钮，加载图像。滑动"全球阈值"的红色条及时调整采样阈值。点击"更新"按钮来检查的效果。 </li><li>勾选"计算中和/滴定"框。点击"采集"按钮以量化的ICP。点击"保存"按钮，在出现提示时保存采样结果。 注:在采样过程后，如果"计算中和/滴定"框中打勾自动出现一个名为"中和滴定与计算"新窗口。 </li><li>负载或输入井-P晚地图。指示阈值（ 例如，50％）"中和:感染扣除（％）"。 </li><li>选择"中和过程"来计算的滴度。检查滴度，并点击"保存并关闭"按钮，保存中和结果。 注:中和被报告为与预定的ICP还原（如50％或80％）对在VC（11栏的图4中的平均值）的血清的最高稀释度的倒数。 50％的减少ICP是在代表性的成果使用。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这项研究中，我们所描述的基于成像的MN测定以量化病毒之间的真实抗原关系。与其它噬斑减少测定法相比，该方法强调测量整个井的ICP，确保感染人口的完全覆盖。其斑块形成的独立性也加宽了测定，以能够形成主要隐形小斑块或只能管理单细胞感染病毒的应用。因此，该测定能够检查更多的病毒和更宽范围的抗体比HI的的影响，并且它有助于更全面地反映抗原相似或病毒12之间的差异。例如，当包括奥塞米韦羧酸盐中，MN测定较少受NA依赖性影响结合，反映抗原的差异更准确。在测定法的发展，大作出了努力以提高实验一致性，速度和检测敏感性，包括通过定量滴定，成像高通量与平板扫描仪输入控制病毒，并实现用户友好的数据处理平台。结果一般都符合并证实了这些HI的。值得注意的是，调查结果还显示所造成的文化选择的或偶发性的变化抗原性改变近期A和B型疫苗病毒抗原漂移变体之间抗原的差异，以及，如在某些A（H1N1）pdm09病毒HA1的G155E替换12。 该协议与相匹配的，给了最强的感染，这大大提高了成像信号的细胞系的选择病毒类型或亚型。实验变异用与测试抗血清的数量平衡重复的设计平滑。的不确定性也可以来自图像质量，这主要是由成像设备的影响。一个体面的彩色平板扫描仪可以显ficantly提高图像的对比度和降低噪音。输入病毒的中和量必须提前定量测定从相应的滴定而病毒仍然保持相似的状态。期间中和，抗血清反应的感染是针对基准病毒（VC）和背景水平（CC）之间的差归一化。 VC和CC的精确测量是中和实验必不可少的。建议更多的重复（一式八份分别在代表性的成果使用）。最后，了解软件是一个实验的成功至关重要。该软件已经过测试，在世卫组织流感中心，英国常规病毒特征进行了两年多。用于处理各种情况的详细说明在补充S1中提供的。 该MN测定法是适合于将样品覆盖一个电话分机的应用remely大视场，但只需要适度的成像分辨率。低成本和简单的设置，使系统随时提供给最病毒学实验室。在相同样品的测量结果的变化小于3％，这大致定义了扫描11中引入的不确定性。这种重复性是在许多病毒学研究的足够，并且可以通过平均从多个扫描图像来进一步降低。 总之，这项研究表明，在抗原性研究中经常使用，并支持目前流通的流感病毒，特别是对病毒列入人类流感疫苗的一年两次的选择详细的分析数据HI强大MN检测。

微中和（MN）测定法在病毒学用于中和抗体的定量和的抗病毒活性。作为血凝抑制的替代（HI）测定，MN测定法可以克服由受体结合于流感病毒的亲和力的改变，其可以变得复杂的HI的解释结果1,2,3-影响无抗原性的影响。直到最近，大多数MN测定法基于细胞病变效应（CPE）或酶联免疫吸附测定（ELISA）4,5。根据焦点和斑块减少MN试验于1990年6,7,8开发的。噬斑减少测定依赖于计数可见斑量化感染。然而，视觉计数仅覆盖大斑是由人眼可分辨的，尽管大多数斑很小，对于许多当前流行的病毒不可见的。这种不完整的覆盖面可能会导致考官之间和实验之间显著的变化，导致我ncomparable结果。另外，也不能使用该方法时，一些病毒表明单个细胞或非常小的斑块流产感染。 计数分辨率差可以通过引入基于成像的协议来改进。随着技术，光学显微镜和高通量的进步孔板读者可以提供准确的方法来计算感染的细胞9。下一个反式照明显微镜，具有标记某些标记感染的细胞可以通过在亚细胞分辨率的吸收或荧光对比度可视化。样品然后可以在电脑屏幕上进行分析。 不幸的是，由于在视场的限制，超过一百平铺图像，需要以覆盖单个孔。判断的板96孔将需要一万左右的图像的摄像处理。这种费力的过程是耗时和昂贵的，并且所获得的分辨率是在属升不必要对病毒感染的常规鉴定。预算有限的实验室可能会发现周围的平板扫描仪内置的方法提供了一种高性价比，高通量的选择。 在本文中，我们描述一种改进的空斑减少测定法MN是适合于大量的病毒的抗原特征和用于定量测定抗病毒活性和中和抗体。该试验具有几个优点:第一，它是一个基于成像的检测，能够测量在细胞水平的病毒感染，无论斑块大小的。内一个井计数总感染的细胞群（ICP）大大增加了检测灵敏度，使得有可能以低感染性表征病毒。其次，更准确地定量滴定之前中和引入到确定输入病毒的量。量化输入病毒显著降低杂物不同的实验之间化，使结果实验室之间更具可比性。第三，中和滴度可以直接通过分析图像，使得定量快速和方便用户的确定。最后，该协议提供了一种具有成本效益和高通量的替代与所需的分辨率和精度。定量是基于一个平板扫描仪和免费数据处理软件。整个安装占地面积小，是大多数实验室部署。 本文提出的协议包括四个主要步骤，包括病毒滴定法，滴定定量，病毒中和，中和定量。病毒滴定是制备实验确定在中和中使用的输入病毒的量。期间滴定，一些病毒浓度在一个96孔板施加到细胞单层。被感染的细胞，然后在第2，病毒定量DILU化，产生20％-85％ICP被依次施加作为输入病毒到相应的中和 第3节中和协议的效价是使用第4节实验证明遵循上述协议是在代表结果提出量化。该法已在过去两年，大多数目前流行的流感病毒，如A（H1N1）pdm09，A（H3N2）和乙型流感病毒彻底的测试。流感病毒鉴定结果已列入给了流感疫苗南半球在2016年和北半球2016至2017年使用建议世界卫生组织协商会议的报告。

<table><tbody><tr><td>VGM</td><td>Sigma</td><td>D6429, P0781</td><td>500 ml DMEM+5 ml Pen/Strep&lt;sup&gt;b&lt;/sup&gt;</td></tr><tr><td>PBS A</td><td></td><td></td><td>Nature pH Phosphate-buffered saline: NaCl 10&amp;nbsp;g, KCl 0.25&amp;nbsp;g, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 1.437 g, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 0.25 g, and Dist. Water 1 L.</td></tr><tr><td>Avicell</td><td>FMC</td><td>RC-581F</td><td>2.4 g in 100 ml distilled water dissolved by agitation on a magnetic stirrer for 1 hr. Sterilize by autoclaving.</td></tr><tr><td>2x DMEM</td><td>Gibco&amp;nbsp;</td><td>21935-028</td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T1426</td><td></td></tr><tr><td>Overlay (10 ml/plate):</td><td></td><td></td><td>5 ml 2x DMEM, Trypsin 2&amp;nbsp;&amp;mu;g/ml final concentration, Avicell 5&amp;nbsp;ml</td></tr><tr><td>Triton X- 100&lt;sup&gt;e&lt;/sup&gt;</td><td>Sigma&amp;nbsp;</td><td>T8787</td><td>Permeabilisation buffer: 0.2% in PBS A (v/v)</td></tr><tr><td>Tween 80</td><td>Sigma</td><td>P5188</td><td>Wash Buffer: 0.05% Tween 80 in PBS A (v/v)</td></tr><tr><td>Horse serum</td><td>PAA Labs Ltd</td><td>B15-021</td><td>ELISA Buffer: 10% in PBS A (v/v) + 0.1% Tween 80</td></tr><tr><td>Mouse MAb against influenza type A</td><td>Biorad</td><td>MCA 400</td><td>1&lt;sup&gt;st&lt;/sup&gt; Antibody: 1:1,000 in ELISA Buffer</td></tr><tr><td>Goat anti-mouse IgG (H+L) HRP conjugate</td><td>Biorad</td><td>172-1011</td><td>2&lt;sup&gt;nd&lt;/sup&gt; Antibody: 1:1,000 in ELISA Buffer</td></tr><tr><td>True Blu peroxidase substrate</td><td>KPL&amp;nbsp;</td><td>50-78-02</td><td>Substrate: True blue +0.03% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;g&lt;/sup&gt; (1:1,000 of 30% solution)</td></tr><tr><td>96-well flat-bottom microtitre plates</td><td>Costar&amp;nbsp;</td><td>3596</td><td></td></tr><tr><td>8 channel multiwall-plate washer and manifold</td><td>Sigma</td><td>M2656</td><td></td></tr><tr><td>Perfection Plate Scanner</td><td>Epson</td><td>V750 Pro</td><td>The imaging software can be downloaded freely from &lt;a href=&quot;http://www.epson.com/cgi-bin/Store/support/supDetail.jsp?oid=66134&amp;amp;infoType=Downloads&quot;&gt;http://www.epson.com/cgi-bin/Store/support/supDetail.jsp?oid=66134&amp;amp;infoType=Downloads&lt;/a&gt;.</td></tr><tr><td>Software operational environment: LabVIEW</td><td>National Instruments Corporation</td><td>Window based with NI Vision builder</td><td>Version: LabVIEW2012 or above</td></tr></tbody></table>

optimization of a quantitative micro-neutralization assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

经上述程序，我们提出最近的抗原特性的实验的一些结果。滴定设置旨在探讨八个输入病毒，与每个稀释度双重复。病毒稀释液进行试验1.0和1.0之间×10 -1×1.0 -6覆盖病毒之间的变化。测试病毒是由H3N2亚型，包括A /喀麦隆/ 15V-二千〇一十五分之三千五百三十八，A /符拉迪沃斯托克/ 36/2015年，A /莫斯科/ 103/2015年，A /托木斯克/ 5/2015 /，A /莫斯科/ 101 / 2015，A /莫斯科/ 100/2015，A /莫斯科/ 133/2015和A /布拉迪斯拉发/ 437/2015。滴定结果示于图5中示出。 图5d说明了的ICP的病毒稀释度的增加而减少。曲线进行归针对的一个井12内产生的所有小区感染同一病毒的ICP（定义为ICP饱和度）。如果ICP并没有随着喜达到饱和ghest病毒浓度，从相应的重复平均ICP代替（A /莫斯科/ 103/2015 图5d）。所产生ICP饱和的30％的病毒稀释液被选择作为输入的病毒稀释度中和（ 表3）。 中和的目的是有一个输入病毒对五种抗血清，对每个板双重复。所示的基准病毒是H3N2 A /斯德哥尔摩/ 63/2015五个抗血清A / HK14分之4801蛋F12 / 15，A / HK十四分之七千二百九十五MDCK F02 / 15，A /南非R2665 / 15 SIAT F50 / 15，A /瑞士十三分之九百七十一万五千九百二十三SIAT NIBF F18 / 15，和A /荷兰十四分之五百二十五SIAT F23 / 15。中和的结果在图6中示出。 图6d示出了具有血清稀释度的增加的感染的进展。在垂直轴上的归一化阳性群体代表的ICP的从对平均ICP相应的抗血清反应的比率的基准病毒12。未感染细胞对照背景ICP正常化过程中扣除。中和滴度被确定为对应于50％的ICP降低（ 表4）的抗血清稀释液的倒数。线性内插来估计滴度两个相邻的血清稀释度之间落下。 <img alt="图1" src="/files/ftp_upload/54897/54897fig1.jpg" /> 图1: 在病毒滴定实验一孔板设置举例。测试病毒被分配到不同的行（A至H）。列是针对不同的病毒稀释液（1到12）。两列被用作复制每个病毒稀释。比例尺= 10毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2: 样品扫描系统的原理图。 （a）在平板扫描仪的成像位置A 96孔板（从再版 附图11用从爱思唯尔BV许可），和（b）中所示的L形位置极限尺寸（一）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54897/54897fig3.jpg" /> 图3:"多孔板阅读器"定量软件的台式图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p&gt; <img alt="图4" src="/files/ftp_upload/54897/54897fig4.jpg" /> 图4: 在中和实验中孔板设置举例。各抗血清被分配给两列重复（1〜10）。行针对不同的血清稀释（A至H）。该病毒控制需要11列，有八个重复（A11至H11）。小区控制是在第12栏，有八个重复（A12至H12）。比例尺= 10毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54897/54897fig5.jpg" /> 图5:滴定实验的结果。 （ 一 ）该井板设置，（ 二 ）扫描孔板图像，（C）说明的颜色编码的，定量，病毒感染的细胞群，和针对病毒稀释液（D）的归一化的病毒感染的细胞群。 30％的ICP用作阈值。 （d）中的误差棒是从样品重复（±1 SD）标准差（SD）。的（b）和（c）比例尺= 10毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54897/54897fig6.jpg" /> 图6: 中和试验的结果。 （ 一 ）该井板设置，（ 二 ）扫描孔板图像，（c）该定量，病毒感染的细胞群的画像，和对血清稀释度（D）的归一化的病毒感染的细胞群。输入VIRUS（VC）是A /斯德哥尔摩/ 63/2015 50％的ICP来计算滴度。 （d）中的误差棒是从样品重复（±1 SD）标准差（SD）。的（b）和（c）比例尺= 10毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/54897fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 补充S1: <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54897/Supplementary_S1.pdf" target="_blank">请点击这里下载该文件。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="3"> 典型的稀释 </td></tr><tr><td></td><td> MDCK细胞 </td><td> MDCK-SIAT1细胞 </td></tr><tr><td> 2天 </td><td> 1:5（1 + 4） </td><td> 1:10（1 + 9） </td></tr><tr><td> 3天 </td&gt; <td> 1:10（1 + 9） </td><td> 1:20（1 + 19） </td></tr><tr><td colspan="3"> 每ml细胞的典型数 </td></tr><tr><td></td><td> MDCK细胞 </td><td> MDCK-SIAT1细胞 </td></tr><tr><td> 2天 </td><td> 2×10 5个 </td><td> 1×10 5个 </td></tr><tr><td> 3天 </td><td> 1×10 5个 </td><td> 5×10 4 </td></tr></tbody></table> 表1:MDCK或MDCK-SIAT1细胞融合瓶的亚文化的典型稀释。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>模式</td><td>专业模式</td></tr><tr><td>文件类型</td><td>膜（该片区指南） </td></tr><tr><td>自动曝光类型</td><td>照片</td></tr><tr><td> IM年龄类型</td><td> 24位彩色</td></tr><tr><td>解析度</td><td> 1200 DPI </td></tr><tr><td>保存的图像格式</td><td> TIFF </td></tr></tbody></table> 表2:平板扫描仪的典型设置。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 行 </td><td> 病毒 </td><td> 建议稀释 </td></tr><tr><td> 一个 </td><td> A /卡梅伦/ 15V-二千〇一十六分之三千五百三十八</td><td> 1.0×10 -2 </td></tr><tr><td> 乙 </td><td> A /符拉迪沃斯托克/2015分之36 </td><td> 1.0×10 -3 </td></tr><tr><td> C </td><td> A /莫斯科/二千〇一十五分之一百〇三</td><td> 1.1×10 -1 </td></tr><tr><td> ð </td><td> A /托木斯克/二千〇一十五分之五</td><td> 5.9×10 -1 </td></tr><tr><td> Ë </td><td> A /莫斯科/二千〇一十五分之一百〇一</td><td> 8.3×10 -1 </td></tr><tr><td> F </td><td> A /莫斯科/二千零十五​​分之百</td><td> 1.4×10 -2 </td></tr><tr><td> G </td><td> A /莫斯科/二千〇一十五分之一百三十三</td><td> 1.3×10 -2 </td></tr><tr><td> H </td><td> A /布拉迪斯拉发/二千〇一十五分之四百三十七</td><td> 1.3×10 -4 </td></tr></tbody></table> 表3:从30％ICP的人口计算的病毒稀释液。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 柱 </td><td> 抗血清 </td><td> 推荐滴度 </td></tr><tr><td> 1 - 2 </td><td> A / HK4801 / 2014 </td><td> 110 </td></tr><tr><td> 3 - 4 </td><td> A / HK7295 / 2014 </td><td> 213.3 </td></tr><tr><td&gt; 5 - 6 </td><td> A /南非/ R2665 / 2015 </td><td> 2155.8 </td></tr><tr><td> 7 - 8 </td><td> A /瑞士/二千〇一十三分之九百七十一万五千二百九十三</td><td> 89.4 </td></tr><tr><td> 9 - 10 </td><td> A /荷兰/二千〇一十四分之五百二十五</td><td> 78.6 </td></tr></tbody></table> 表4:A /斯德哥尔摩的50％ICP /二千零十五分之六十三反对抗血清滴度。

Watch this Scientific Journal Video about Optimization of a Quantitative Micro-neutralization Assay at JoVE.com

Optimization of a Quantitative Micro-neutralization Assay

这项研究描述了一个 成像基于微中和测定来分析病毒之间的抗原关系。该协议采用平板扫描仪，有四个步骤，包括滴定，滴定定量，中和，中和定量。该法与目前流行的流感A（H1N1）pdm09，A（H3N2）和乙型流感病毒效果很好。

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

optimization-of-a-quantitative-micro-neutralization-assay

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

21.1K 次观看.  The Francis Crick Institute. 这项研究描述了一个 成像基于微中和测定来分析病毒之间的抗原关系。该协议采用平板扫描仪，有四个步骤，包括滴定，滴定定量，中和，中和定量。该法与目前流行的流感A（H1N1）pdm09，A（H3N2）和乙型流感病毒效果很好。 

视频: Optimization of a Quantitative Micro-neutralization Assay

使用合成寡核苷酸的 DNA 酶检测

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

作者聚焦：表征来自极端微生物和常见病原体的新型酶以了解 DNA 修复和复制

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

使用修饰的合成寡核苷酸检测核酸代谢酶

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to ...

科研

生物化学

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 ...

免疫与感染

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

细胞法检测酶替代疗法中细胞吸收的抗体

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

生物学

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

全自动毛细 Electrophoretic-based 免疫分析方法的程序和关键优化策略

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

使用基于比色细胞的测定法检测针对 AAV 的中和抗体的分步方法

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

定量微中和试验的优化

摘要

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