墨尔本世界卫生组织合作中心的参考与研究流行性感冒是由健康的澳大利亚政府部门的支持。

1.确定荧光产物4-MU的一个荧光的线性范围<ol><li>通过在5mL无水乙醇中溶解11.3毫克4-MU的制备10毫升6.4毫4-MU原液。加入5毫升0.9％的NaCl（W / V）到原液，使10毫升。通过在1×毫升9测定缓冲液（33.3毫2-（N-吗啉代）乙磺酸（MES）和4mM CaCl 2的，pH 6.5）中稀释1毫升6.4毫4-MU的制备640μM的工作溶液。 注意:通过将13克MES和8 2至992的1M的CaCl毫升毫升蒸馏水准备2X测定缓冲液中。调整使用NaOH的10 M中的pH值至6.5。过滤使用孔径为0.2μm的无菌醋酸纤维素过滤器的缓冲区。警告！氢氧化钠是腐蚀性，并可能导致化学灼伤皮肤和眼睛。确保全套个人防护装备佩戴。 </li><li>制备一系列4-MU浓度（ 即，5μM，10μM，20μM，40μM，80μM，160＆＃的5mL的181; M和320μM）通过使用1×测定缓冲液中640μM4-MU的2倍系列稀释。 </li><li>分配4-MU（ 即 ，5μM，10μM，20μM，40μM，80μM，160μM，320μM和640μM）（每个稀释两个孔）成清晰，96的各系列稀释的50μL的孔平底板和50μL1×测定缓冲液中的进入剩余孔（其作为空白以测量背景信号）。 注意:在该反应体积中的最终浓度4-MU（50μL4-MU + 50μL300μMMUNANA）是2.5μM，5μM，10μM，20μM，40μM，80μM，160μM和320μM 。 </li><li>通过在20毫升的蒸馏水重构25毫克MUNANA的制备的2.5mM MUNANA储备溶液。混合0.72毫升2.5姆·芒南的用1x毫升5.28测定缓冲液以获得300μMMUNANA工作溶液（足够的体积为一个板）。覆盖含有300μMMUNANA干活的管用铝箔克溶液，并保持在冰上，除非立即使用。丢弃任何剩余的材料。 注:2.5mM的MUNANA储备溶液可以储存在-20℃下1个月，并且必须一个冷冻/解冻循环内使用。 4-MU和MUNANA解决方案是光敏感的，必须从长时间曝光的保护。 </li><li>添加300μMMUNANA50μL的每个孔中，轻轻拍打以混合，并在37℃下温育30分钟。盖上板密封，以防止水分蒸发的板块。 注意:此步骤是考虑到在NA抑制测定背景荧光。 </li><li>通过混合11毫升无水乙醇中以2.225毫升0.824 1M的NaOH的（足够的体积为一个板）准备终止溶液。 </li><li>加入100μL终止液至每个孔以停止反应，并轻轻拍打混合。 </li><li>读取使用具有355纳米的激发波长设定和460nm的发射波长设置荧光计板中，如PEr是制造商的说明。 </li><li>计算使用来自不含有4-MU的所有孔的荧光信号的平均背景信号。减去各含有4-MU的孔的该平均背景信号并计算平均信号（RFU）对于每个4-MU浓度。绘制RFU的标准曲线针对4-MU浓度（μM），如在图1a中示出;曲线的特写线性截面示于图1b。 </li><li>可视化RFU的针对4-MU浓度（μM），以确定线性范围和最佳的目标信号的曲线图;线性范围是，其中按比例的荧光信号增加至4-MU上的情节的浓度的增加，而最佳的目标信号是在线性范围内的任意的4-MU浓度。 注:线性范围和最佳的目标信号是荧光计特定的。例如，在墨尔本WHOCCRRI荧光计具有2的线性范围0.5-40μM4-MU和30〜μM4-MU，其对应于〜1500 RFU的最佳目标信号。 </li></ol> 2.确定病毒的NA活性注:流感病毒培养至在将Madin-Darby犬肾（MDCK）细胞或胚鸡蛋8足够滴度。 <ol><li>分配每未稀释的培养流感病毒的阱120μL到塔1和1x测定缓冲液的60微升含0.1％NP-40到96孔，U型底的板的其余11列。 </li><li>串联横跨板进行的病毒两倍稀释（ 即，从第1列转移60微升至第2列，等等，直至第11列），使用多道移液器，离开塔12仅含有1×测定缓冲液空白。 </li><li>从各孔中（稀释病毒和空白）的转移50μL的成清晰，96孔，平底板上。 注:这是没有必要到c焊割移液管尖端，如果材料被从塔12通过传递到第1列。 </li><li>添加300μMMUNANA 50μL（按照步骤1.4中制备）每孔并轻轻拍打板以混合。孵育在37℃下将板1个小时。盖上板密封，以防止水分蒸发的板块。 </li><li>添加100μL的终止溶液（按照步骤1.6中制备）每孔中以终止反应，并轻轻拍打板以混合。 </li><li> 阅读使用荧光板。 <ol><li>使用的355纳米的激发波长设定和460nm的发射波长设置。 </li></ol></li><li>确定基于在列12中的荧光读数平均背景信号和从各孔中减去平均背景信号。绘制RFU针对病毒稀释度的曲线图。 注意:在WHOCCRRI墨尔本100μMMUNANA背景值典型地50和120之间的RFU，但是这些将根据荧光计贝不同纳克使用。 </li><li>查看针对病毒稀释液RFU的曲线，以确定每个病毒（ 图2）的曲线的线性部分的中点。使用最佳的目标信号（在步骤1中确定的）作为基准点。 注意:这应与在第1确定的荧光计的4-MU线性范围对应，并且将提供在部分3中使用的病毒的适当的浓度。 </li></ol> 3.评估病毒易感性的NA抑制剂使用NA抑制测定<ol><li> 在300μM的浓度制备NA抑制剂的主股。 <ol><li>由5.0毫克扎那米韦溶解于2×毫升50分析缓冲液（66.6毫MES和8毫米氯化钙2，pH 6.5）中制备300μM的扎那米韦（分子量，MW =332.32克/摩尔）。 </li><li>通过在50ml 2×试验缓冲液的5.8毫克溶解;制备300μM奥塞米韦羧酸盐（MW =386.44克/摩尔D-酒石酸盐）。 </li><li>准备300μM帕拉米韦三水合物（MW =382.45克/摩尔）通过在50ml 2×试验缓冲液的5.7毫克溶解。 </li><li>通过在50ml 2×试验缓冲液的5.2毫克溶解制备300μM拉尼米韦（MW =346.34克/摩尔）。 注意:NA抑制剂主股可以储存在-20℃下12个月。检查NA抑制剂的MW以确保正确的重量和体积都在复原中使用。的奥塞米韦羧酸盐是前药的磷酸奥司他韦的活性化合物。因此，只有奥塞米韦羧酸盐应在NA抑制试验中使用。 </li></ol></li><li>从主股票，准备在0.03纳米，0.3纳米，3纳米，30纳米，300纳米，3000纳米的浓度工作在50mL离心管中的NA抑制剂的10倍系列稀释的股，和30000纳米的2X测定缓冲液（66.6毫MES和8毫米氯化钙2，pH 6.5）中;这是一个跨多个试验使用。 注意:在该反应volum的最终浓度NA抑制剂E（病毒稀释+ 50 NA的μL抑制剂+ 50 300μMMUNANA的μL的50μL）是0.01纳米，0.1纳米，1纳米，10纳米，100nm时，1000纳米，和10,000纳米，分别。的最终浓度不包括100微升的终止溶液。存储在2-8℃NA所有抑制剂稀释液。到期日期是相同主股。 </li><li>制备在1×测定缓冲液中的病毒稀释液含有在96深孔块0.1％NP-40表面活性剂。使用基于从部分2得到的NA活性测定结果病​​毒稀释使用每病毒2毫升的总体积，以测试4 NA抑制剂。 注:准备每病毒两个孔（每孔1毫升）在96深孔块。病毒稀释液进行病毒1，2，和3中的实例示于表1中 。 </li><li>分配NA抑制剂（按照步骤3.2中制备）的所需要的体积为8深井水库。从那里，在稀释范围分配50 NA抑制剂μL从0纳米（2X测定B仅uffer）〜30000nm的行A至H中一个明确的，96孔，平底板上。 注:板布局在图3中示出。 </li><li>每孔加入50微升的稀释的测试病毒的列1-11和每1×测定缓冲液中的孔50μL只塔12轻轻敲击板以混合，并在室温下孵育45分钟。盖上板密封，以防止水分蒸发的板块。 </li><li>添加300μMMUNANA 50μL（按照步骤1.4中制备）每孔并轻轻拍打板以混合。孵育在37℃下将板1个小时。盖上板密封，以防止水分蒸发的板块。 </li><li>添加100μL的终止溶液（按照步骤1.6中制备）到每个孔中，并轻轻拍打板以混合。 </li><li> 阅读使用荧光板。 <ol><li>使用的355纳米的激发波长和460nm的发射波长，如先前所描述。 </li></ol></li></ol> 4.计算IC50值注:JASPR V1.2是曲线拟合软件，使IC 50值的计算。该软件是由流感司在疾病预防控制中心，美国亚特兰大开发的。该软件利用等式:V = V 最大 ×（1 - （[I] /（K I + [I]））），其中V max是代谢的最大速率，[I]是抑制剂浓度，V是该响应被禁止，和K i是抑制曲线的IC 50。 <ol><li>复制并粘贴由荧光计输出到一个12塔板的格式（96孔）电子表格中的原始数据，从单元格A1。 注:原从每个后续板的数据必须通过一个空行错开。如果每个病毒已被针对4种NA抑制剂测试，粘贴一组四个的原始数据（与每个板之间的空行）。 </li><li>打开安装软件和CLICK的"实验"选项卡上。选择"替代2药物氟11个样品。" </li><li>单击"选项"选项卡，并勾选"生成图"。 </li><li>再次点击"选项"，然后单击"新建项"选项卡上。保存"inhibition_key" .csv文件在同一文件夹中的原始数据表格文件。 </li><li>在inhibition_key文件，列出以下ID的所有样品名称。每个样品名称必须是唯一的。 </li><li>如果四个NA抑制剂进行了测试，插入空格下面奥塞米韦两个附加行，键入"帕拉米韦"和"拉尼米韦"。保存到inhibition_key文件所做的更改。 </li><li>返回到"jaspr 1.2抑制曲线拟合"窗口，选择原始数据文件作为"实验文件"和inhibition_key的"密钥文件"。运行分析。保存为.csv和.pdf格式的结果。 注:该软件会自动绘制抑制曲线（RFU agaiNST NA抑制剂浓度），如示于图4。该程序还计算针对个体NA抑制剂（ 图4）的每种病毒的IC 50值。此外，JASPR呈现IC 50值和信号对背景（S / B）比率在电子表格的格式。背景值可以从实验室取决于所使用的荧光计的不同而不同实验室。在墨尔本WHOCCRRI，背景为100μMMUNANA范围内的值从50至120 RFU。一个可靠的IC 50值，的一个≥10S / B比是优选的，虽然小于10的比率是仍可接受的，特别是对于具有非常低的NA活性的突变病毒。 </li><li>检查IC 50值和由软件产生的曲线的形状。所有的数据点应落在或靠近曲线;如果他们不这样做，重复NA抑制试验。 注:对于病毒显示异常高的IC 50值，试验应重复ED确认结果。 </li></ol>

<ol>
	<li>Moscona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuraminidase+inhibitors+for+influenza.">Neuraminidase inhibitors for influenza.</a> N.Engl.J.Med. 353 (13), 1363-1373 (2005).</li><li>Hurt, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+update+on+the+susceptibility+of+human+influenza+viruses+to+neuraminidase+inhibitors.">Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors.</a> Antiviral Res. 132, 178-185 (2016).</li><li>Meijer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+update+on+the+susceptibility+of+human+influenza+viruses+to+neuraminidase+inhibitors.">Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors.</a> Antiviral Res. 110, 31-41 (2014).</li><li>Takashita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+update+on+the+susceptibility+of+human+influenza+viruses+to+neuraminidase+inhibitors.">Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors.</a> Antiviral Res. 117, 27-38 (2015).</li><li>Lackenby, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+resistance+to+oseltamivir+among+influenza+A(H1N1)+viruses+in+Europe.">Emergence of resistance to oseltamivir among influenza A(H1N1) viruses in Europe.</a> Euro Surveill. 13 (5), (2008).</li><li>Hurt, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community+transmission+of+oseltamivir-resistant+A(H1N1)pdm09+influenza.">Community transmission of oseltamivir-resistant A(H1N1)pdm09 influenza.</a> N Engl J Med. 365 (26), 2541-2542 (2011).</li><li>Takashita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+large+cluster+of+influenza+A(H1N1)pdm09+viruses+cross-resistant+to+oseltamivir+and+peramivir+during+the+2013-2014+influenza+season+in+Japan.">Characterization of a large cluster of influenza A(H1N1)pdm09 viruses cross-resistant to oseltamivir and peramivir during the 2013-2014 influenza season in Japan.</a> Antimicrob Agents Chemother. 59 (5), 2607-2617 (2015).</li><li>Eisfeld, A. J., Neumann, G., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+virus+isolation,+culture+and+identification.">Influenza A virus isolation, culture and identification.</a> Nat Protoc. 9 (11), 2663-2681 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meetings+of+the+WHO+working+group+on+surveillance+of+influenza+antiviral+susceptibility+-+Geneva,+November+2011+and+June+2012.">Meetings of the WHO working group on surveillance of influenza antiviral susceptibility - Geneva, November 2011 and June 2012.</a> Wkly Epidemiol Rec. 87 (39), 369-374 (2012).</li><li>Okomo-Adhiambo, M., Hurt, A. C., Gubareva, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemiluminescent+neuraminidase+inhibition+assay:+a+functional+method+for+detection+of+influenza+virus+resistance+to+the+neuraminidase+inhibitors.">The chemiluminescent neuraminidase inhibition assay: a functional method for detection of influenza virus resistance to the neuraminidase inhibitors.</a> Methods Mol Biol. 865, 95-113 (2012).</li><li>Hurt, A. C., Okomo-Adhiambo, M., Gubareva, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescence+neuraminidase+inhibition+assay:+a+functional+method+for+detection+of+influenza+virus+resistance+to+the+neuraminidase+inhibitors.">The fluorescence neuraminidase inhibition assay: a functional method for detection of influenza virus resistance to the neuraminidase inhibitors.</a> Methods Mol Biol. 865, 115-125 (2012).</li><li>Analysis of IC50 data. isirv Antiviral Group (isirv-AVG) Available from: <a target="_blank" href="https://isirv.org/site/index.php/methodology/analysis-of-ic50-data">https://isirv.org/site/index.php/methodology/analysis-of-ic50-data</a> (2016)</li><li>Sleeman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R292K+substitution+and+drug+susceptibility+of+influenza+A(H7N9)+viruses.">R292K substitution and drug susceptibility of influenza A(H7N9) viruses.</a> Emerg Infect Dis. 19 (9), 1521-1524 (2013).</li><li>Gubareva, L. V., Robinson, M. J., Bethell, R. C., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+and+framework+mutations+in+the+neuraminidase+active+site+of+influenza+viruses+that+are+resistant+to+4-guanidino-Neu5Ac2en.">Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en.</a> J Virol. 71 (5), 3385-3390 (1997).</li></ol>

作者什么都没有透露。

流感病毒易感性NA抑制剂的全球监测当前正在通过使用荧光或化学发光NA抑制测定11,12许多实验室进行。荧光测定法比化学发光测定更常用。虽然这两个测定法是健壮和可再现的，从基于荧光的测定中获得的IC 50值通常比基于化学发光的测定法更高，使得来自两个试验难以13的数据直接比较。即使使用相同的协议的，从一个实验室产生的数据可以从另一个会有所不同。由于实验室之间的这些变化，世卫组织工作组抗流感病毒敏感性监测生产的指导方针，以协助实验室间的比较。而不是比较绝对IC 50值，该指南使用基于IC 50倍的差异，以中位数在每个特定的实验室测试的NI流感病毒的IC 50 SA比较。比较从五个协作中心数据的能力已导致全球流感抗病毒药物敏感性数据的2，3，4年出版。在公共领域的大量流感易感数据的可用性，使研究人员从与在自己的实验室中产生的研究将IC 50个数据。 即采取类似的概念之外NA抑制实验也可购得。包含准备使用的试剂，这些商业试剂盒（抑制剂不包括NA）同样可重复性。然而，在内部NA抑制测定比商业试剂盒基本上便宜，因为大多数所用试剂可以在内部中进行较大的量和MUNANA衬底，其先前由该测定的主要成本，现在可以从各种源竞争力的价格购买。每种药物测试一个流感分离物的成本大约是$ 1（USD）。在墨尔本WHOCCRRI，做了改进，以在内部NA抑制测定的机器人平台的掺入用于测定的液体处理组件之后。除了病毒稀释度的手动制备，大部分的程序所使用的处理液体的机械臂来执行。这不仅最大限度地减少人工处理，但同时也增加了可在一天内运行试验的数量。 虽然NA抑制测定是高度可靠，有许多的需要用额外的小心完成关键步骤。首先，在NA抑制剂浓度可以转移抑制曲线和IC 50值的任何不规则性;因此，应小心谨慎制备NA抑制剂浓度时支付。其次，准确吸取和精确的潜伏期是保持整个检测结果的一致性至关重要;这可以通过使用校准的移液管和定时器来实现。控制病毒在每次试验中包括也使的检测性能从试验到检测，并在很长一段时间的监控。第三，由于季节性流感病毒的NA的酶活性是在pH 6.5最佳，测定缓冲液的正确的pH是重要的。一些报道已经发现，使用较低的pH条件下可以改善流感变体，如含有R292K突变14,15 A（H7N9）变体的鉴定。然而，修改至测定缓冲液的pH将转向的IC 50值，这可能实验室内和实验室之间数据的比较复杂化。其他的修改和故障排除，可以是performed列于表5中 。 该NA抑制剂是唯一批准的类抗病毒药物是目前对抗循环流感病毒有效。直到其他抗病毒类临床使用变得可用，流行的流感病毒的抗病毒易感性的监视将仅集中在NA抑制剂。由于简单性和结果的再现性，使用NA抑制试验，以评估流感病毒易感性的NA抑制剂将继续。

血细胞凝集素（HA）和神经氨酸酶（NA）是A型流感和B型流感病毒的两种主要表面糖蛋白。 HA结合到细胞表面的糖蛋白或糖脂的唾液酸半乳糖，而NA通过从细胞表面1上的半乳糖裂解唾液酸释放病毒。的NA抑制剂是一类被合理设计紧密结合到酶NA活性位点，从而防止病毒后代的释放和传播流感的抗病毒药物。奥司他韦和扎那米韦是已批准在全球许多国家用于治疗和流感预防2 NA抑制剂。近年来，两个额外的NA抑制剂帕拉米韦和拉尼米韦，已被批准为国家数量有限的用途。的流感病毒对易感性的NA抑制剂和赋予抗性突变的鉴定筛选是在确定和监测有效实现了重要这个类抗病毒药物的eness。 在过去16年中，基于荧光的NA抑制测定已被常规地在WHO进行合作中心参考与研究流感，墨尔本（墨尔本WHOCCRRI）监测抗病毒易感性之间循环流感病毒的变化趋势。每年，超过2000种流感病毒对抗病毒药物敏感性测试。在大多数流感季节，所述病毒&gt; 98％易受所有四个NA抑制剂2，3，4中 ，虽然在2007-2008北半球流感季节，有在原季节性A（H1N1）病毒的数量猛增这减少了敏感性奥司他韦5。这组病毒，其含有NA氨基酸取代H275Y，传播到世界的由2008年底的其余部分，使得奥塞米韦inappropri全球吃了这种病毒的治疗。绝大多数当前流行的流感B，流感A（H3N2）和流感A（H1N1）pdm09菌株易受奥塞米韦，虽然含有NA氨基酸取代H275Y赋予减少奥塞米韦A（H1N1）pdm09变体的社区簇和帕拉米韦易感，已报道在世界6,7的各个部分。 因为需要有一个足够高的病毒滴度，临床标本（包括动物鼻洗液）必须在任一细胞培养物进行传代或含胚前的抗病毒敏感性测试鸡卵。此文章中描述的NA抑制测定法可分为三个部分: 确定线性范围上的特定荧光计荧光产物4-甲基伞形酮（4-MU） 由于荧光计之间的固有差异，叔他的线性范围为荧光终产物，4-MU，相对荧光单位（RFU），需要建立。一旦4-MU的线性范围建立，最佳目标信号被选择，向其中的流感病毒的浓度在NA活性测定进行调整。一旦完成了特定的荧光，这不应该需要重复。 确定病毒的NA活性 的NA活性测定是一个简单的测定，其涉及添加2&#39;的- （4-甲基伞形酮）-α-D-Ñ-acetylneuraminic酸（MUNANA）衬底以连续稀释病毒。从MUNANA由NA裂解产生的荧光终产物4-MU量被使用荧光计测量。适当的病毒稀释度在NA抑制测定使用由针对病毒稀释绘制荧光单元中选择。从所产生的S形曲线，直线部的中点应该对应于在第1确定的荧光计的4-MU的线性范围和将通知的病毒的适当的浓度在第3节中使用。 评估病毒易感性使用NA抑制测定NA抑制剂 为了评估的病毒到特定NA抑制剂的敏感性，在第2节中所确定的稀释的病毒温育与一系列NA抑制剂浓度的。以下与MUNANA随后的温育后，通过未抑制病毒产生的4-MU在RFU通过荧光计测定。上的病毒的NA酶活性的NA抑制剂的抑制效果是根据以减小NA活性的50％所需的NA抑制剂浓度，给出作为IC 50值来计算。

<table><tbody><tr><td>Influenza A and B viruses</td><td></td><td>Cultured in MDCK cells or 9 day old embryonated specific pathogen free (SPF) eggs</td><td></td></tr><tr><td>Madin-Darby Canine Kidney (MDCK) cells</td><td>ATCC</td><td>PTA-6500</td><td></td></tr><tr><td>2-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA)&amp;nbsp;</td><td>Biosynth AG</td><td>M-5507</td><td></td></tr><tr><td>2-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA)&amp;nbsp;</td><td>Sigma</td><td>M8639</td><td></td></tr><tr><td>4-Methylumbelliferone (4-MU)</td><td>Sigma</td><td>M1381-25G</td><td></td></tr><tr><td>2-[N-morpholino]ethanesulphonic acid (MES hydrate) (free acid)&amp;nbsp;</td><td>Sigma</td><td>M8250-250G</td><td></td></tr><tr><td>Calcium Chloride (Ca Cl&lt;sub&gt;2&lt;/sub&gt;)&amp;nbsp;</td><td>APS AJAX Finechem&amp;nbsp;</td><td>127-500G</td><td></td></tr><tr><td>Surfactant-Amps-NP-40 (10% solution)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>PIE28324</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)&amp;nbsp;</td><td>APS AJAX Finechem</td><td>482-2.5KG</td><td></td></tr><tr><td>Absolute Ethanol&amp;nbsp;</td><td>APS AJAX Finechem</td><td>214-2.5L GL</td><td></td></tr><tr><td>96-well clear flat-bottom plates</td><td>NUNC</td><td>456537</td><td></td></tr><tr><td>96-well U-bottom plates</td><td>Greiner Bio-one&amp;nbsp;</td><td>4650101</td><td></td></tr><tr><td>8 channel deep well block</td><td>Pacific Laboratory Products</td><td>RES-MW8-HP</td><td></td></tr><tr><td>96-well deep plates, 2.0 mL square wells</td><td>Pacific Laboratory Products</td><td>P-2ML-SQ-C</td><td></td></tr><tr><td>Plate sealers</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>236366</td><td></td></tr><tr><td>Bottle-top vacuum filter system (cellulose membrane (nitrate), pore size 0.2 &amp;mu;m, membrane area 33.2 cm&lt;sup&gt;2&lt;/sup&gt;, filter capacity 500 mL)</td><td>Sigma-Aldrich</td><td>CLS430758-12EA</td><td></td></tr><tr><td>Single-channel pipettes (1 &amp;micro;L - 1,000 &amp;micro;L)</td><td>Variety of suppliers (eg. Eppendorf, Sartorius)</td><td></td><td></td></tr><tr><td>Multi-channel pipettes</td><td>Variety of suppliers (eg. Eppendorf, Sartorius)</td><td>8 or 12 channel electronic and manual pipette (5 - 1250 &amp;micro;L volume)</td><td></td></tr><tr><td>Pipette tips (1 &amp;micro;L - 1,250 &amp;micro;L)</td><td>Variety of suppliers (eg. Eppendorf, Sartorius)</td><td></td><td></td></tr><tr><td>Disposable pipettes (10 mL and 25 mL)</td><td>Greiner Bio-one&amp;nbsp;</td><td>P7740-200EA and P7865-200EA</td><td></td></tr><tr><td>Pipette controller</td><td>Eppendorf</td><td>4430000018</td><td></td></tr><tr><td>Centrifuge tubes 50 mL</td><td>BD Bioscience</td><td>352070</td><td></td></tr><tr><td>Racked tubes</td><td>Scientific Specialties, Inc.</td><td>1750-00</td><td></td></tr><tr><td>Fluorometer with excitation wavelength setting of 355 nm and an emission wavelength setting of 460 nm</td><td>TermoFisher Scientific</td><td>ASCENT FL 374</td><td></td></tr><tr><td>Ascent software</td><td>TermoFisher Scientific</td><td>5185410CD</td><td></td></tr><tr><td>Incubator set at 37 &amp;deg;C</td><td>Lab Supply</td><td>Biocell 1000</td><td></td></tr><tr><td>Zanamivir</td><td>GlaxoSmithKline</td><td>Request directly from the company</td><td></td></tr><tr><td>Oseltamivir carboxylate</td><td>Roche</td><td></td><td></td></tr><tr><td>Peramivir (BCX-1812)</td><td>BioCryst</td><td></td><td></td></tr><tr><td>Laninamivir (R-125489)</td><td>Daiichi-Sankyo</td><td></td><td></td></tr><tr><td>&amp;nbsp;JASPR v1.2</td><td>Influenza Division at the CDC Atlanta, USA&amp;nbsp;</td><td>freely available upon request (fluantiviral@cdc.gov)</td><td></td></tr></tbody></table>

fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza viruses to the neuraminidase inhibitor class of antivirals

神经氨酸酶（NA）抑制剂是唯一的类批准用于那些对目前流行株有效的流感的治疗和预防的抗病毒药物。除了它们在治疗季节性流感的使用过程中，NA抑制剂已被一些国家在大流行的情况下，使用的储存。据监测循环流感病毒这一类抗病毒药物的敏感性是非常重要的。有不同类型的可用于评估的流感病毒的NA抑制剂的敏感性测定的，但使用一个荧光底物或化学发光底物的酶抑制测定法是最广泛使用的，并建议。这个协议描述了使用基于荧光的测定法，以评估NA抑制剂流感病毒的易感性。该测定法是基于NA酶裂解2&#39; - （4-甲基伞形酮）-α-D-Ñ-acetylneuraminic酸（MUNANA）衬底以释放荧光产物4-甲基伞形酮（4-MU）。因此，基于给定为IC 50值，该值是必需的，以减少NA活性的50％的NA抑制剂的浓度，确定对流感病毒NA安·娜抑制剂的抑制效果。

从世界卫生组织工作组抗流感病毒易感性9的监控用标准化的报告准则，的流感病毒的NA抑制剂的敏感性使用的术语正常抑制（NI），降低的抑制（RI），和抑制高度还原的报道（HRI） 。 NI病毒是那些与IC超过10倍以下50个值相比，中值IC 50为参考A型流感病毒（或小于5倍为乙型流感病毒）。 RI病毒是那些具有10和100倍平均IC 50的基准为A型流感病毒（或5-和50倍的流感B）以上之间IC 50值。 HRI病毒是那些与IC中值IC 50的基准为A型流感病毒（或以上50倍为乙型流感病毒）100倍的50个值以上; 见表2。 50个值被计算，每年在墨尔本WHOCCRRI更新，以反映的IC 50值的微小变化循环的流感病毒株的NA抑制剂（ 表3）。流感A（H1N1）的中位IC 50值pdm09病毒是在四个NA抑制剂几乎相同，但中间扎那米韦和拉尼米韦IC为A（H3N2）病毒50倍的值是2至4倍相比奥司他韦和帕拉米韦IC 50个值（ 表3）。对B型流感病毒的中位数奥塞米韦IC 50值通常为5至10倍比扎那米韦，培拉米韦，和拉尼米韦IC 50值（ 表3）高。 的NA抑制测定是表型分析，不提供关于遗传变化associ信息ated与RI或HRI。因此，重要的是，遗传分析是以下的用RI或HRI病毒的鉴定进行。在墨尔本WHOCCRRI，变体的NA基因，使用Sanger测序和焦磷酸测序分析。的，可以在与RI和HRI变体病毒的NA基因中找到的氨基酸取代的代表性例子示于表4中 。可以改变NAI易感性的氨基酸替换一个更广泛的名单也可在世卫组织网站10。 <img alt="图1" src="/files/ftp_upload/55570/55570fig1.jpg" /> 图1: 针对4-MU浓度RFU。 （a）中针对4-MU浓度（μM）的RFU的标准曲线。虚线框示出了4-MU的用于荧光计的线性范围。线性范围以上的荧光信号可以是饱和的，并且因此，任何小号在荧光商场的变化可能不被荧光计来检测。 （b）中图1a的用于的识别标准曲线的特写线性部分"最佳目标信号"。在墨尔本WHOCCRRI荧光计具有2.5-40μM4-MU的线性范围和〜30μM4-MU的最佳目标信号，其对应于〜1500 RFU。 <a href="http://ecsource.jove.com/files/ftp_upload/55570/55570fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55570/55570fig2.jpg" /> 图2: 流感病毒的NA活性曲线的实施例。 50.61 RFU的平均背景值已从在NA活性曲线每次稀释点减去。箭头指示病毒适当稀释在NA抑制测定每种病毒的使用。为了方便准备病毒稀释液aration，可以选择用于病毒3代替的1/96稀释进行1/100稀释。 <a href="http://ecsource.jove.com/files/ftp_upload/55570/55570fig2large.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/55570/55570fig3.jpg" /> 图3: 板布局为NA抑制测定的设置。每个板包括最后一列，其充当不包含病毒，但仅1×测定缓冲液（AB），NA抑制剂，MUNANA阴性对照，和终止液。注:JASPR使用读数从每块板的第12列以确定IC 50值的计算中使用的平均空白信号。 <a href="http://ecsource.jove.com/files/ftp_upload/55570/55570fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图4: 一个A（H1N1）病毒pdm09，A /珀斯/ 82/2015 的抑制曲线和IC 50 值 的实施例 。该JASPR软件呈现针对NA抑制剂浓度的增加（nM）的抑制曲线作为荧光（RFU），所述曲线中的每个点拟合。基于抑制曲线上，则IC 50值确定为NA抑制剂的浓度，以减少病毒NA活性的50％。 <a href="http://ecsource.jove.com/files/ftp_upload/55570/55570fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>病毒</td><td>需要稀释病毒</td><td> 1X测定缓冲液体积（μL） </td><td>表面活性剂的安培-NP-40（10％）（mL）的</td><td>病毒体积（mL） </td></tr><tr><td> 1 </td><td> 1/20 </td><td> 940 </td><td> 10 </td><td> 50 </td></tr><tr><td> 2 </td><td> 1/40 </td><td> 965 </td><td> 10 </td><td> 25 </td></tr><tr><td> 3 </td><td> 1/100 </td><td> 890 </td><td> 10 </td><td> 10 </td></tr></tbody></table> 表1:病毒稀释液中的NA抑制测定用于制备病毒1，2，和3。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2">病毒类型/子类型/谱系</td><td>普通抑制</td><td>抑制减少</td><td>高度降低的抑制</td></tr><tr><td> （NI） </td><td> （RI） </td><td> （HRI） </td></tr><tr><td> A（H1N1）pdm09 </td><td> &lt;10倍</td><td> 10-100倍</td><td> &gt; 100倍</td></tr><tr><td> A（H3N2） </td><td> ＆＃60; 10倍</td><td> 10-100倍</td><td> &gt; 100倍</td></tr><tr><td>乙山形和B维多利亚</td><td> &lt;5倍</td><td> 5-50倍</td><td> &gt; 50倍</td></tr></tbody></table> 表2:WHO抗病毒药工作组建议指引流感病毒易感性的NA抑制剂的分类。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2">病毒类型/子类型/谱系</td><td rowspan="2"> ñ </td><td>扎那米韦</td><td>奥司他韦</td><td>帕拉米韦</td><td>拉尼米韦</td></tr><tr><td>中位数（范围）IC 50 nM的</td><td>中位数（范围）IC 50 nM的</td><td>中位数（范围）IC 50 nM的</td><td>中位数（范围）IC 50 nM的</td></tr><tr><td> A（H1N1）pdm09 </td><td> 1326 </td><td> 0.42（0.1-3.43） </td> &lt;TD&gt; 0.36（0.01-3.48） </td><td> 0.19（0.07-1.60） </td><td> 0.55（0.05-2.29） </td></tr><tr><td> A（H3N2） </td><td> 1654 </td><td> 0.9（0.11-4.0） </td><td> 0.38（0.01-3.65） </td><td> 0.33（0.12-3.06） </td><td> 1.38（0.01-9.38） </td></tr><tr><td>乙山形和B维多利亚</td><td> 1115 </td><td> 2.2（1.24-10.72） </td><td> 15.12（2.39-70.75） </td><td> 1.36（0.57-6.67） </td><td> 2.89（1.62-9.15） </td></tr></tbody></table> 表3:中值IC 50 和IC 50 范围内的正常的抑制（NI）从2015年病毒在WHO CCRRI，墨尔本的。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2">氨基酸取代</td><td rowspan="2">类型/亚型/谱系</td><td colspan="4"> IC 50的倍数变化共mpared参考中值IC 50值。 </td></tr><tr><td>扎那米韦</td><td>奥司他韦</td><td>帕拉米韦</td><td>拉尼米韦</td></tr><tr><td> H275Y </td><td> A（H1N1）pdm09 </td><td> 1 </td><td> 557（HRI） </td><td> 123（HRI） </td><td> 2 </td></tr><tr><td> E119V </td><td> A（H3N2） </td><td> 1 </td><td> 63（RI） </td><td> 1 </td><td> 1 </td></tr><tr><td> H134Y </td><td>乙维多利亚</td><td> 1 </td><td> 4 </td><td> 76（HRI） </td><td> 2 </td></tr><tr><td> N151T </td><td>乙维多利亚</td><td> 4 </td><td> 4 </td><td> 42（HRI） </td><td> 1 </td></tr><tr><td> G104E </td><td>乙维多利亚</td><td> 1220（HRI） </td><td> 87（HRI） </td><td> 17724（HRI） </td><td> 701（HRI） </td></tr><tr><td> E105K </td><td>乙维多利亚</td><td> 3 </td><td> 5（RI） </td><td> 59（HRI） </td><td> 2 </td></tr><tr><td> I222T </td><td>乙维多利亚</td><td> 2 </td><td> 7（RI） </td><td> 8（RI） </td><td> 3 </td></tr><tr><td> H273Y </td><td>乙山形</td><td> 1 </td><td> 230（HRI） </td><td> 377（HRI） </td><td> 2 </td></tr><tr><td> D197N </td><td>乙山形</td><td> 4 </td><td> 7（RI） </td><td> 32（RI） </td><td> 3 </td></tr></tbody></table> 表4:链接到降低抑制（RI）或高度还原的抑制（HRI），以NA抑制剂氨基酸取代代表清单。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>问题</td><td>可能的原因（S） </td><td>解决方案（S） </td></tr><tr><td rowspan="2">无或低的NA活性</td><td>没有病毒存在或低病毒产量。 </td><td>临床标本必须在细胞系中进行培养（ 即将Madin-Darby犬肾细胞）或在含胚鸡卵到在NA抑制测定使用较高的病毒负荷。 </td></tr><tr><td>一些突变病毒具有非常低的NA活性，尽管在高病毒载量。 </td><td>用整齐的病毒浓度进行测试。可以使用较低的pH值的测定缓冲液（ 例如 pH 5.3）。然而，在比较数据时一定要小心。 </td></tr><tr><td rowspan="3">否或在NA抑制测定低NA活性</td><td>不加入病毒。 </td><td>再稀释病毒。确保病毒被直接加入到1×测定缓冲液中。 </td></tr><tr><td>在使用了错误的病毒稀释。 </td><td>重复NA活性测定。 </td></tr><tr><td>没有足够的孵化时间。 </td><td>确保孵育时间之后。 </td></tr><tr><td rowspan="3">数据点落在IC 50曲线外</td><td rowspan="2">较高浓度的NA抑制剂交叉污染。 </td><td>确保提示是不与NA INHIBI接触TOR分配稀释病毒到96孔板中时。 </td></tr><tr><td>如果使用一个8深井水库，丢弃和重新分配NA抑制剂浓度到一个新的8深井水库。 </td></tr><tr><td>的NA抑制剂或MUNANA或稀释的病毒的体积不是同样加入到每个孔中。 </td><td>重复用校准的多通道移液器测定。确保每个试剂的等体积分配到每个孔中。 </td></tr><tr><td rowspan="3">异常高的IC 50值</td><td>加入病毒的浓度过高。 </td><td>重复NA活性测定和NA抑制测定。 </td></tr><tr><td>测试样品含有流感A和流感B的混合物</td><td>进行实时PCR，以确定病毒混合物的存在。 </td></tr><tr><td>样品中的细菌污染</td><td>与抗生素存在的无菌条件下培养病毒。 </td></tr><tr><td rowspan="2">高背景荧光信号</td><td> MUNANA衬底可以随时间降解。 </td><td>使用新一批MUNANA subtrate的。 </td></tr><tr><td>从邻近井的荧光检测。 </td><td>使用黑色96孔平底板</td></tr></tbody></table> 表5:在NA抑制测定潜在的问题进行故障排除。

Watch this Scientific Journal Video about Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals at JoVE

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

我们描述了使用表型的基于荧光的神经氨酸酶抑制测定的到的甲型和乙型流感病毒的易感性评估对神经氨酸酶抑制剂类抗病毒药物。

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 ...

fluorescence-based-neuraminidase-inhibition-assay-to-assess

Research

JoVE Journal

Immunology and Infection

17.7K 次观看.  Peter Doherty Institute for Infection and Immunity. 我们描述了使用表型的基于荧光的神经氨酸酶抑制测定的到的甲型和乙型流感病毒的易感性评估对神经氨酸酶抑制剂类抗病毒药物。 

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

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

在调节Toll样受体激活Neu1唾液酸酶活动的检测

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

科研

免疫与感染

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

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, ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

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 ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

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.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

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. ...

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 ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

高通量抗病毒检测筛查寨卡病毒复制抑制剂

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Source: Gao, J., et al. <a href="https://app.jove.com/v/54573">Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.</a> J. Vis. Exp. (2016).
This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

一种测量流感神经氨酸酶抑制抗体滴度的测定法

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and ...