This project was funded by intramural PanFlu funds from the Center for Biologics Evaluation and Research (CBER). Plasmids used to prepare reassortant viruses were kindly provided by Robert Webster, St Jude Children's Research Hosptial, Memphis, TN. We are indebted to Ewan Plant and Haruhiko Murata for critical review of the manuscript.

禽流感的元器件均重配病毒，必须使用生物安全2级（BSL2）的批准，由农业部美国农业部（USDA）和生物安全委员会使用的实验室 - 增强的做法来处理。 1.试剂和起始材料的制备注:请参阅材料表 ，以获得所有试剂的来源。 <ol><li>胎球蛋白包被的板<ol><li>通过混合用90ml10毫升10X包被缓冲液制备涂覆缓冲液的去离子H 2 O </li><li>制备胎球蛋白在25毫克/毫升，在500微升等份1X包被缓冲液并贮存在-20℃的母液。 </li><li>通过稀释原液1000倍在1×涂层缓冲器准备立即涂装板前胎球蛋白（25微克/毫升）的工作溶液。 </li><li>使用多道移液器以分配100μl的胎球蛋白的工作溶液到人具有高蛋白质结合能力的96孔平板l的孔中。 </li><li>盖上板密封各板，然后堆叠在板中的10组，并在箔包裹它们。 </li><li>进行检测之前将平板在冰箱（2-8℃）至少18小时。 注意:将板可以涂覆预先并存储在2-8℃的涂覆溶液达2个月。 </li></ol></li><li>稀释剂<ol><li>以制备稀释液，使病毒和样品稀释液（2-（N-吗啉代）乙磺酸（MES），pH为6.5，20mM的氯化钙 ，1％牛血清白蛋白（BSA）和0.5％吐温20），混合94.2毫升1X MES，用2ml的CaCl 2（10毫克/毫升）pH为6.5，3.3毫升30％的BSA和0.5毫升吐温20。 </li><li>以制备用于PNA-HRPO（MES，用20mM的CaCl 2和1％BSA pH6.5）中的稀释液，用2毫升氯化钙 （10毫克/毫升）混合94.7毫升1×MES，pH 6.5的，和3.3毫升30％BSA的。 </li></ol></li><li>神经氨酸酶（NA）的 注:H6Nx病毒reassortants，产生（这些具有H6亚型HA和感兴趣的NA）的下列公开的方法5,10。从合作者请求灭活重组病毒的小瓶，如果他们不是在内部使用。当使用灭活的病毒，确认该制剂适合于通过示范在ELLA使用该灭活过程并没有对NA活性的显著影响。 <ol><li>培养H6Nx病毒在鸡胚11股票和存储整齐尿囊液以0.5ml等份在-80℃。 </li><li>使用病毒的新等分试样用于每个试验。不要冻结和解冻的等分。 </li></ol></li><li>血清样品和对照<ol><li>热灭活所有血清（测试样品如前和接种后的血清，以及作为对照， 例如 ，样品与已知的NI滴度）的在56℃水浴中45-60分钟。前或热处理后储存在-20至-70℃的血清。如果SAMP莱要重复测试，冷冻，以使样品不重复冷冻和解冻之前做出几个等分试样。 </li><li>使用ELLA测量是在合理的可供数量（&gt; 5毫升）的人血清的NI滴度，以确定至少一个与低滴度和另一个具有高滴度。存储100微升等份作为≤-20℃，以使相同的样品可以用作以跟踪测定性能在许多测定法的对照。 </li></ol></li><li>花生凝集素（PNA） - 辣根过氧化物酶（HRPO） <ol><li>制备PNA-HRPO稀释剂（MES，用氯化钙2和1％BSA pH6.5）中。 </li><li>通过在1ml稀释剂的1毫克的PNA-HRPO溶解制备PNA-HRPO原液。存放于-20°C 20-200微升等分。 </li><li> 500，1:750 1:1000和1:使用新的PNA-HRPO很多，测试前1 2000稀释该试剂，以确定导致最大外径与阳性对照的量的（病毒只）和背景（没有病毒）笔帽是&lt;阳性信号的10％。 <ol><li>作出2.1节所述一式四份病毒稀释。 </li><li>转移稀释到胎球蛋白包被的板作为在第2.2节中所述，在37℃下孵育板。 </li><li> 16-18小时后洗涤板，并添加1:500 1:750 1:1000和1:2000稀释的PNA-HRPO（100微升/孔）到复制板的行。 </li><li>如步骤2.3.4所述2.3.9完成检测。 </li><li>查看数据来选择，导致了&gt; 10倍背景的最大信号稀释。 </li></ol></li><li>在使用前，立即准备PNA-HRPO的最佳稀释1.5.3鉴定。 </li></ol></li><li>制备大体积的洗涤缓冲液（0.01M磷酸缓冲盐水（PBS），pH值7.4，0.05％吐温20（PBS-T））的。存放在室温下。 </li><li>准备过氧化物酶底物（邻苯二胺二盐酸盐（OPD））:注:另一种过氧化物酶底物如3,3&#39;，5,5&#39;-四甲基联苯胺（TMB），也可以使用<ol><li>通过在试验当天溶解在100毫升的dh 2 O 1胶囊制备磷酸盐-柠檬酸盐缓冲液。 </li><li>使用前立即溶解在20毫升的磷酸盐柠檬酸盐缓冲液的1 OPD片剂（10毫克）。 </li></ol></li><li>制备终止溶液（1 NH 2 SO 4）27.2 ml储备98％的H 2添加SO 4至973毫升卫生署2 O.混合，然后在室温保存。 </li></ol> 2. NA量的测定中使用ELLA <ol><li>制备病毒稀释在96孔平板<ol><li>免除120微升样品稀释液（1.2节）为列稀释板1-11。 </li><li>以第1列，增加一个额外的96微升样品稀释液。 </li><li>解冻，然后加入24微升复制在第1列孔中这给出1:10稀释病毒的前涡旋病毒的小瓶中。 </li><li>使样品稀释病毒的系列2倍稀释液，通过转移120微升从一个井每个稀释度下一次使用干净的枪头。 </li></ol></li><li>转移病毒稀释到胎球蛋白包被的板<ol><li>用PBS-T（1.6节）洗胎球蛋白包被的板3次，然后吸干各板放置吸水纸巾以除去任何过量的洗涤缓冲液。 </li><li> 1添加50微升样品稀释液（1.2.1节）向每孔中的列的胎球蛋白包被的板11。 </li><li>加入样品稀释液列12 100微升（这些井都是阴性对照）。 </li><li>转印稀释的病毒的50微升从列稀释板1-11的胎球蛋白包被的板的相应孔中。 </li><li>盖上板密封该板，然后将其放置在湿润的培养箱中在37℃。 </li><li>记的在这余下的步骤将被执行（16-18小时后）的时间。 </li></ol></li><li>完成NA测定<ol><li>当孵育完成后（16-18小时），转移板块在板凳上，取下板盖。 </li><li>用PBS-T（1.7节）洗涤该板6次，然后反转和在吸水纸巾轻拍，以确保所有液体已经被从孔中除去。 </li><li>添加100μl/孔的PNA-HRPO溶液（在1.5.3中确定的稀释度）至所有孔中，并孵育该板在RT 2小时。 </li><li>不到15分钟前的孵化结束，如1.7节的介绍准备OPD解决方案。 </li><li>洗试验板3次，以除去PNA-HRPO并加入100微升OPD底物的向每个孔之前吸干。 </li><li>将培养板在RT恰好10分钟。 </li><li>停止加入100微升/ 1N的硫酸孔中的反应。 </li><li>使用平板读数器测量490nm处的光密度（OD值）为0.1秒。 </li><li>保存并导出所有数据文件。 </li></ol></li><li>选择将用于血清学检查病毒稀释<ol><li>画绘出OD 490nm处的图</sUB&gt;在每个病毒稀释度值。检查滴定曲线来确定的最大信号（这通常是对应于高原在滴定曲线的开始的信号），最小的信号（即包含在板的12列中没有病毒提供背景对照孔），和病毒的导致在外径是正比于输入稀释（ 即，曲线的线性区域）的稀释液。 </li><li>选择该病毒稀释度给出了最大信号的约90％，并且是线性的范围内。确认在所选择的稀释的外径为至少10倍于背景信号大。使用选定的病毒稀释于采用这种特殊的病毒股票的所有检测。 注意:可替换地，测量病毒的NA活性和在ELLA使用〜15-20μUNA活性/毫升。 </li></ol></li></ol> 3.酶联凝集素测定法注: 图2笑WS稀释和测定板的设置。 <ol><li>使样本稀释<ol><li>放置在冰上的热灭活样品。 注:8血清可以在每个板中稀释。 </li><li>对于设置使用在开始各板块全线1:10稀释，再加入120微升样品稀释液中列3-11所有孔。 </li><li>到第2列，加样品稀释液216微升，每个样品的24微升。通过上下吹打3次在混合井的样品，然后转移120μl到下一列。 </li><li>改变枪头后，上下吹打混合井的内容，然后转移120μl到下一列。 </li><li>重复步骤3.1.4直到样品已被转移到塔11，剩余的120微升丢弃。 </li></ol></li><li>加样品及病毒的胎球蛋白涂层板<ol><li>解冻病毒，涡流的小瓶中并悬浮病毒在于在步骤2中选择的稀释稀释剂（1.2节）。4.2。至少准备5毫升病毒为每个测定板。保持稀释的病毒在冰上，直到洗涤板和血清样品已被添加到该板。 </li><li>决定所需要的用于测定胎球蛋白包被的板的数量（一般也适用每板4的血清）。用PBS-T洗胎球蛋白包被的板3次，然后反转每个板并吸干到吸水纸巾以除去过量的洗涤缓冲液。 </li><li>使用多道移液器，从稀释板转移50微升每个血清控制或样品稀释成复孔列2-11。 </li><li>加入50μl稀释的病毒的所有孔，除了阴性对照组（第12列）。 </li><li>加入50微升样品稀释液对井1列，并添加100微升样品稀释液中列12。 </li><li>盖上板密封的孔，然后通过轻轻拍打板的侧面，或在中等速度放置在板振荡器上持续10秒混合。 </li><li>放置板在一个humidif在37℃下16-18小时IED孵化器。 </li></ol></li><li>添加PNA-HRPO并完成试验在2.3节中描述</li></ol> 4.数据分析<ol><li>确定化验结果的有效性<ol><li>确认该背景值（无病毒）是阳性对照（病毒和无血清）的小于10％。 </li><li>确认对照血清运行在不同的测定使用相同的条件下，效价中的中位数滴度的2倍。 </li><li>确认控制井的OD测量是一致的（≤20％不同），并一式两份样品孔的OD测量是一致的（≤10％不同）。 </li><li>确定无效结果的根本原因并重复检测，如果在4.1.1，4.1.2或4.1.3所列的标准，都没有得到满足。考虑尝试解决在表1中的因素。 </li></ol></li><li>分配50％的终点效价<ol><li>对于每块测定板，苏btract从所有读数的平均背景（无抗原加入孔）。 </li><li>计算每个血清稀释度的抑制百分比用以下公式:100×（OD 病毒唯一的控制 - OD 测试样品 ）/ OD 病毒唯一的控制 。 </li><li>确定导致最大信号的至少50％抑制的最高稀释度。 </li><li>举报此稀释为50％终点效价的倒数。 注意:如果50％抑制完全没有任何稀释实现的，效价是低于测试的第一稀释， 例如 ，&lt;10时1:10是测试的第一次稀释。 </li></ol></li><li>计算50％抑制浓度（IC 50） <ol><li>使用四参数逻辑回归来确定IC 50滴度如下:减去所有读数的平均背景，然后将结果传送到执行回归分析的程序。 </li><li>使用非线性回归，与最大设定该病毒仅控制和最小设置为零，以确定与恰好50％抑制对应的稀释度。 </li><li>报告该稀释作为IC 50滴度的倒数。 </li></ol></li></ol>

<ol>
	<li>Kilbourne, E. D., Laver, W. G., Schulman, J. L., 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=Antiviral+activity+of+antiserum+specific+for+an+influenza+virus+neuraminidase.">Antiviral activity of antiserum specific for an influenza virus neuraminidase.</a> J Virol. 2 (4), 281-288 (1968).</li><li>Compans, R. W., Dimmock, N. J., Meier-Ewert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antibody+to+neuraminidase+on+the+maturation+and+hemagglutinating+activity+of+an+influenza+A2+virus.">Effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza A2 virus.</a> J Virol. 4 (4), 528-534 (1969).</li><li>Schulman, J. L., Khakpour, M., Kilbourne, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+specific+immunity+to+viral+neuraminidase+on+influenza+virus+infection+of+mice.">Protective effects of specific immunity to viral neuraminidase on influenza virus infection of mice.</a> J Virol. 2 (8), 778-786 (1968).</li><li>Murphy, B. R., Kasel, J. A., Chanock, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+serum+anti-neuraminidase+antibody+with+resistance+to+influenza+in+man.">Association of serum anti-neuraminidase antibody with resistance to influenza in man.</a> N Engl J Med. 286 (25), 1329-1332 (1972).</li><li>Sandbulte, M. R., Gao, J., Straight, T. M., Eichelberger, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miniaturized+assay+for+influenza+neuraminidase-inhibiting+antibodies+utilizing+reverse+genetics-derived+antigens.">A miniaturized assay for influenza neuraminidase-inhibiting antibodies utilizing reverse genetics-derived antigens.</a> Influenza Other Respi Viruses. 3 (5), 233-240 (2009).</li><li>Lambre, C. R., Terzidis, H., Greffard, A., 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=Measurement+of+anti-influenza+neuraminidase+antibody+using+a+peroxidase-linked+lectin+and+microtitre+plates+coated+with+natural+substrates.">Measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates.</a> J Immunol Methods. 135 (1-2), 49-57 (1990).</li><li>Couzens, L., 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=An+optimized+enzyme-linked+lectin+assay+to+measure+influenza+A+virus+neuraminidase+inhibition+antibody+titers+in+human+sera.">An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera.</a> J Virol Methods. 210C, 7-14 (2014).</li><li>Eichelberger, M. 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=Comparability+of+neuraminidase+inhibition+antibody+titers+measured+by+enzyme-linked+lectin+assay+(ELLA)+for+the+analysis+of+influenza+vaccine+immunogenicity.">Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity.</a> Vaccine. 34 (4), 458-465 (2016).</li><li>Westgeest, K. B., 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=Optimization+of+an+enzyme-linked+lectin+assay+suitable+for+rapid+antigenic+characterization+of+the+neuraminidase+of+human+influenza+A(H3N2)+viruses.">Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses.</a> J Virol Methods. 217, 55-63 (2015).</li><li>Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., 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=A+DNA+transfection+system+for+generation+of+influenza+A+virus+from+eight+plasmids.">A DNA transfection system for generation of influenza A virus from eight plasmids.</a> Proc Natl Acad Sci U S A. 97 (11), 6108-6113 (2000).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Animal Influenza Diagnosis and Surveillance. , (2002).</li><li>Cate, T. R., 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=A+high+dosage+influenza+vaccine+induced+significantly+more+neuraminidase+antibody+than+standard+vaccine+among+elderly+subjects.">A high dosage influenza vaccine induced significantly more neuraminidase antibody than standard vaccine among elderly subjects.</a> Vaccine. 28 (9), 2076-2079 (2010).</li><li>Couch, R. B., 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=Antibody+correlates+and+predictors+of+immunity+to+naturally+occurring+influenza+in+humans+and+the+importance+of+antibody+to+the+neuraminidase.">Antibody correlates and predictors of immunity to naturally occurring influenza in humans and the importance of antibody to the neuraminidase.</a> J Infect Dis. 207 (6), 974-981 (2013).</li><li>Fritz, R., 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=Neuraminidase-Inhibiting+Antibody+Response+to+H5N1+Virus+Vaccination+in+Chronically+Ill+and+Immunocompromised+Patients.">Neuraminidase-Inhibiting Antibody Response to H5N1 Virus Vaccination in Chronically Ill and Immunocompromised Patients.</a> Open Forum Infect Dis. 1 (2), (2014).</li><li>Fries, L. F., Smith, G. E., Glenn, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombinant+viruslike+particle+influenza+A+(H7N9)+vaccine.">A recombinant viruslike particle influenza A (H7N9) vaccine.</a> N Engl J Med. 369 (26), 2564-2566 (2013).</li><li>Monto, A. S., 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=Antibody+to+Influenza+Virus+Neuraminidase:+An+Independent+Correlate+of+Protection.">Antibody to Influenza Virus Neuraminidase: An Independent Correlate of Protection.</a> J Infect Dis. 212 (8), 1191-1199 (2015).</li><li>Fritz, R., 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=A+vero+cell-derived+whole-virus+H5N1+vaccine+effectively+induces+neuraminidase-inhibiting+antibodies.">A vero cell-derived whole-virus H5N1 vaccine effectively induces neuraminidase-inhibiting antibodies.</a> J Infect Dis. 205 (1), 28-34 (2012).</li></ol>

The authors have no conflicts of interest.

酶联凝集素测定法是测量血清NI抗体滴度的实用方法。虽然ELLA被Lambre 等人 ，于1990年描述的，其接受为标准的血清学测定法已更近，与众多的实验室进行试验以测量临床样品12-16中的NI抗体滴度。一些修改可以使协议步骤，而不产生很大的影响，以所测量的NI滴度。例如，PBS可以用作稀释剂，然而，这是非常有益的，比较在推荐缓冲液（MES，pH6.5）中和病毒滴定曲线的PBS之前作出决定，因为一些NA亚型显著降低酶的活性在pH值&gt; 7.0。当不使用的最佳pH，病毒的最大信号可能会降低，导致使用在测定抗原（ 即病毒）的过量的;在这些条件下，该测定可以具有降低的敏感性。 一些无当未处理的血清在ELLA进行测试，观察特定正 - 抑制。这是通过热处理（56℃45分钟）除去，这表明耐热β-抑制剂的存在。其它非特异性抑制剂（α和γ级）流感HA的已被描述，特别是涉及到的H3N2病毒血凝和感染性。的确，非特异性抑制，可以在ELLA观察时H3N2病毒被用作NA的来源;这种抑制被处理的血 ​​清样品与唾液酸酶9少量除去。 至于其他的血清学测定法，阴性和阳性对照应包括在每个试验提供评估测定性能的装置。当测定没有达到验收标准，原因应该被识别。 表1提供了可寻址到解决问题在测定潜在的步骤的列表。 该协议ELLA省本报告中被提供的使用包含来自禽流感病毒，房委会发起重配病毒，NA的来源。这就出现了一个限制，因为许可证需要具有低致病性禽流感病毒的工作进行了检测。 H6Nx重配病毒可实验室之间共享它们已被灭活后。因此该测定可通过协作进行一次实验室产生重组病毒已证明灭活完成并且准备保留了其的NA活性。使用H6Nx重配病毒的约束可以通过使用NA的非感染性的来源来克服。例如，一些实验室已经使用纯化的重组NA 17，而其他人已经使用的病毒样颗粒（VLP）15。然而，无论是重组NA也不病毒样颗粒都是现成的，因此，我们继续使用流感病毒抗原性不匹配HA禽流感。 传统法测量的NI抗体滴度不几个原因实用;最令人关注的是，需要以产生颜色反应的有害化学物质。此外，需要样品的大体积和测定是麻烦的执行。与此相反，ELLA容易进行并且是一个格式，允许样品的合理数目的滴定。从研究的数据，审查ELLA变化表明，试验产量可重复的结果7,8。的ELLA也随之由Ni抗体滴度可能的常规测量，并且可以预料它会被实验室进行血清学研究更频繁地使用。 有迹象表明，需要在执行ELLA时，需要考虑几个关键步骤。首先，NA活性的非特异性抑制剂必须被移除。这些抑制剂通常是不耐热和45分钟在56℃下通过热处理被破坏。热处理是足以除去非特异性FIC从样品抑制剂H6时重配病毒被用作NA的来源，然而，当H3N2病毒在ELLA使用时，结合于血凝素血清因素也干扰了ELLA并应通过用唾液酸酶的少量去除热处理9之前。第二，抗原， 即 ，重配H6Nx病毒过量的，不应该被用作这降低该测定的灵敏度。因此病毒仔细滴定是一个必不可少的步骤;抗原在每个测定中使用的量必须提供一个信号，该信号以及上述背景，并且必须是滴定曲线， 即线性范围内时，信号（光密度）必须是成比例的病毒稀释度。 除了常规的血清学，ELLA的可用于评估的季节性流感病毒的NA抗原之间的差异。对列入选择的病毒株时，此信息可能会非常有用小号候选疫苗，并会方便一点导致NA抗原漂移的免疫压力更深入的了解。另外，从猪或禽流感病毒的NA抗原性分析可以决定新菌株的大流行潜力提供关键信息。

神经氨酸酶（NA）为流感病毒颗粒的表面上表达的糖蛋白。其酶活性是用于从感染细胞1,2-新形成的病毒颗粒的释放是至关重要的。抑制NA活性的抗体减少病毒滴度和疾病症状的动物模型3和对人类疾病的抵抗力4关联。因此在NA抑制（NI）的滴度接种后的增加可以用作疫苗的功效的一个指标，然而，许多过去的流感免疫原性研究没有包括这个终点，因为传统的测定法来测量的NI抗体滴度是不切实际的常规血清学。 测量的NI滴度的传统方法是基于量化是从糖缀合物由NA 1切割的唾液酸量。这种方法，通常被称为硫代巴比妥酸（TBA）的方法，使用危险化学品的转化唾液酸交流id来，可以通过光谱法进行定量的发色团。该法是不适合，因为用于各个玻璃管检测大量的样品，使得该测定繁琐。该试验在96孔板的微型化提供了一种更实用的格式如图5所示 ，但是该测定仍然需要使用有毒的化学品，因此是不理想的。 备选测定法来测量的NI抗体滴度通过Lambré 等 6开发的。该测定通过测定糖蛋白，半乳糖的倒数第二糖，当唾液酸为NA释放其变成露出的量量化的酶活性。由于花生凝集素（PNA）特异性结合半乳糖，一个PNA - 辣根过氧化物酶（HRPO）偶联物可用于获得比色读出。被测量的光密度因此正比于样品中的NA活性。 NI滴度通过确定的最高稀释度测的血清抑制与NA活性的至少50％。此酶联凝集素测定法（ELLA）被涂覆有胎球蛋白，一种高度糖基化的血清蛋白的96孔板中进行，作为基板为NA。 用于测量的NI滴度测定法的一个重要的考虑，是不适用的来源。这是因为从接种或感染的个体的血清含有抗体血凝素（HA），以及抗体的NA。结合于HA的抗体可以与NA活性干扰，因此，为了避免由HA特异性抗体的非特异性抑制，纯化的NA或全病毒含有抗原不匹配的HA应在测定中使用。这些病毒可以通过经典的重配或反向遗传学生成;目标是营救其中包含不相关的目标应变一个亚型的HA病毒，和NA是正在研究的病毒。在这篇文章中描述的测定采用这是由REV产生A型流感病毒ERSE遗传学含有H6亚型HA和有针对性的NA由A型流感病毒，H1N1亚型和H3N2 5。 由ELLA所产生的结果和微型TBA试验表明这些方法相媲美，具有相似的亚型的特异性和灵敏度7。 ELLA不需要使用危险化学品，因此是测量的NI滴度的首选方法。这是很容易进行，只用几个步骤（ 图1）:样品稀释并转移到添加了胎球蛋白包被的板到病毒（NA的来源）。将板孵育O / N和添加的PNA-HRPO之前洗涤。 2小时温育后，将板洗涤并添加过氧化物酶底物;显色反应停止并最终光密度测定和样品的稀释，导致NA活性的至少50％抑制的倒数被报告为50％终点滴度。一个内部实验室研究，以评估reproducibili的ELLA的TY，表明板到板的变异是最小的，操作者对操作者重复性导致滴度7不超过2倍的差异。 ELLA变异随后实验室间的研究表明，在不同的实验室进行时和列入标准可以进一步减少结果8变异测定重复性好。的ELLA是适于从临床前和临床流感疫苗研究7测定血清面板NI抗体滴度并且还可以用来评价流感病毒9在NAS之间抗原性的差异。

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">coating buffer&#160;</td>
			<td style="width:267px;">KPL</td>
			<td style="width:113px;">50-84-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;fetuin</td>
			<td>Sigma</td>
			<td>F3385</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5X MES, pH 6.5&#160;</td>
			<td>KD-Medical</td>
			<td>PBS-0134</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma</td>
			<td>C7902</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% BSA</td>
			<td>Sigma</td>
			<td>A8327</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin PNA-HRPO</td>
			<td>Sigma</td>
			<td>&#160;L7759</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS-T</td>
			<td>Sigma</td>
			<td>P3563-10PAK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">o-Phenylenediamine dihydrochloride</td>
			<td>Sigma</td>
			<td>P8287</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate-citrate buffer</td>
			<td>Sigma</td>
			<td>P4922</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sulfuric acid</td>
			<td>Sigma</td>
			<td>258105</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well&#160; Maxisorp plates</td>
			<td>NUNC</td>
			<td>439454</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well round bottom well plates</td>
			<td>NUNC</td>
			<td>267245</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate sealers</td>
			<td>Thermo Scientific&#160;</td>
			<td>14-245-192B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chicken eggs</td>
			<td>Charles River Laboratories</td>
			<td>9 day old embryonated specific pathogen free (SPF) chicken eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">multichannel pipette</td>
			<td>Variety of suppliers e.g., Rainin, Eppendorf</td>
			<td>8 or 12 channel manual pipettem 50-250 &#181;l volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette tips</td>
			<td>Depends on pipettor brand</td>
			<td>Depends on pipettor brand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader, with 490 nm filter</td>
			<td>Perkin Elmer</td>
			<td>Victor V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">water bath set to 37 &#176;C</td>
			<td>Variety of suppliers&#160;</td>
			<td>Variety of models</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">water bath set to 56 &#176;C</td>
			<td>Variety of suppliers&#160;</td>
			<td>Variety of models</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">refrigerator set to 4 &#176;C</td>
			<td>Variety of suppliers&#160;</td>
			<td>Variety of models</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">incubator set to 37 &#176;C</td>
			<td>Variety of suppliers&#160;</td>
			<td>Variety of models</td>
		</tr>
	</tbody>
</table>

measuring influenza neuraminidase inhibition antibody titers by enzyme-linked lectin assay

抗体神经氨酸酶（NA），对流感病毒的第二个最丰富的表面蛋白，对预防流感的保护作出贡献。传统的方法来测量NA抑制剂（NI）抗体滴度不适合常规血清学实用。这个协议描述酶联凝集素测定法（ELLA），来测量在涂覆有一个大的糖蛋白底物，胎球蛋白96孔板中进行的NI滴度一个实用的替代方法。 NA裂解末端唾液酸从胎球蛋白，露出倒数第二糖，半乳糖。花生凝集素（PNA）是具有特异性的半乳糖和因此desialylation的程度的凝集素可以使用PNA-辣根过氧化物酶缀合物，随后加入生色过氧化物酶底物进行定量。被测量的光密度成比例的NA活性。为了测量NI抗体滴度，血清的连续稀释液在37℃孵育CO / N与固定数额Ø胎球蛋白涂层板˚FNA。的最高血清稀释度，其导致的NA活性≥50％抑制的倒数被指定为NI抗体滴度。该ELLA为人类抗体反应例行评估以下流感感染或疫苗接种的实用格式。

在这个手稿提交的测定在图1示意示出。 图2示出了96孔板的布局，并表示该血清样品的连续稀释液在稀释板中制备它们转移到胎球蛋白包被的板之前。该测定的一个重要参数是在测定中使用的神经氨酸酶的量;这是通过重配病毒的滴定法测定。 H6N1和H6N2病毒滴定的例子示于图3中 。在H6N1 1:10，1:20和1:40的稀释液，光学浓度是相似的，表明条件是得到最大读数。在1:80稀释，读出的是最大的约90％，因此，在随后的测定中，使用该稀释病毒。血清滴定的例子示于图4。该图显示，这是一个人的血清样品的TItrated对抗H6N1和H6N2病毒。每个数据点表示是由血清的连续稀释液来实现的NA活性的抑制百分率;在H6N1检测的第一个血清稀释度是1:80;在H6N2测定血清的第一稀释度为5。因为其导致≥50％抑制的最高稀释度是NI抗体滴度，在本实施例中所示的血清的效价为640对数控的NA / 99（N1）和160针对WI的NA / 05（N 2）。 <img alt="图1" src="/files/ftp_upload/54573/54573fig1.jpg" /> 图 1. 的ELLA协议的示意性抗原（病毒）的稀释液（A）的测定在测定（该协议的步骤2）使用:病毒稀释液加入到胎球蛋白包被的平板和NA活性通过测定通过PNA-HRPO结合量化末端半乳糖残基在协议步骤2.3描述; （B </stron<html> NI血清抗体滴度（协议的第3步）克&gt;）测定。样品稀释，然后转移至其中添加病毒胎球蛋白包被的板。该板O / N将PNA-HRPO和完成生成显色反应所需的步骤之前培养。最后，显色反应停止，光密度测量。样品稀释导致的NA活性的至少50％的抑制率的倒数报告为50％终点效价。 <a href="https://www.jove.com/files/ftp_upload/54573/54573fig1large.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/54573/54573fig2.jpg" /> 图2.图显示ELLA板成立。血清样品的系列稀释液在稀释板制成，然后转移到一个胎球蛋白包被的板。<html> 3fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54573/54573fig3.jpg" /> （蓝色符号/布里斯班/显示二千零七分之五十九（H1N1）A的NA）和H6N2 UR / 07（A娜H6N1和H6N2病毒滴定 图3. 例子。H6N1 BR / 07系列稀释/乌拉圭/ 716 / 2007（H3N2）的红色符号表示） 孵育在胎球蛋白包被的平板18小时，并用PNA-HRPO如上所述确定的反应性。平均 2孔的OD 490nm处绘制在病毒稀释度的倒数。选择用于在ELLA使用H6N1的稀释度为1:80和H6N2的选择的稀释度1:40因为这些稀释液造成的最大光密度的约90％，并且是线性范围内。3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54573/54573fig4.jpg" /> 图4. 对N1的NA（红色符号）和N2血清（蓝色符号）亚型的滴定。对重组病毒测定NA的抑制滴度H6N1 NC / 99（包含A的NA /新喀里多尼亚/ 20 /日（H1N1） ）和H6N2 威斯康星/ 05（包含A的NA /威斯康星/二千零五分之六十七（H3N2））。血清的系列稀释液百分之酶抑制被示出具有指示50％抑制水平虚线。针对A的NAS上的NA的抑制滴度/新喀里多尼亚/ 20 /日（H1N1）（NC / 99）和A /威斯康星/二千零五分之六十七（H3N2）（WI / 05）分别为2 9.3（640）和2 7.3（ 160），分别<a href="https://www.jove.com/files/ftp_upload/54573/54573fig4large.jpg" target="_blank">请点击这里查看大图已经</a>这个数字rsion。 <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） </td><td> 解 </td></tr><tr><td>信号弱或没有在病毒滴度达到平台期</td><td>我）低NA酶活性 ⅱ）病毒原液不是最佳的条件下储存</td><td> ⅰ）确认该稀释剂具有pH值是最佳的NA活性;如果pH值是最优的，准备一个新的病毒股票或集中病毒 II）再生和分装股票;干冰单元小瓶冷冻在-80℃下储存前</td></tr><tr><td>弱或无颜色阳性细胞对照孔</td><td>我）病毒稀释错误分配 II）瓶到瓶变异病毒冷冻等分 III）PNA- HRPO变性或稀释的太多 四）OPD准备不正确</td><td>一世）;重复病毒滴度 ⅱ）从相同批次滴定几个小瓶，以确保不存在变异性。如果显著变化，准备好新鲜等分 三）使用最佳的病毒稀释返滴定PNA- HRPO ⅳ）用OPD的新鲜制剂重复</td></tr><tr><td>弱或无抑制的阳性对照血清</td><td> ⅰ）在测定中使用过多的病毒 二）血清恶化</td><td>我）重复病毒滴度 ii）取得新的抗血清检查储存条件</td></tr><tr><td>抑制作用阴性对照血清</td><td>血清I）不足热处理 ⅱ）在测定中使用太少病毒</td><td> ⅰ）重复血清热灭活 II）重复病毒滴度</td></tr><tr><td>高背景</td><td>我）板可能污染 II）PNA- HRPO浓度过高/过低</td><td>我）重复使用新鲜的涂层板 II）滴定PNA-HRPO，以确定正确的稀释使用</td></tr><tr><td>病毒滴定示出了在低稀释度的NA活性的表观抑制</td><td>我）尿囊液可能含有NA基板</td><td> ii）该已经通过蔗糖垫沉淀使用病毒</td></tr></tbody></table> 不符合性能标准测定的表1中根本原因分析 。此表提供了可能的原因和解决方案在执行时，ELLA可能出现的问题。

Watch this Scientific Journal Video about Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay at JoVE.com

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

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 ...

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Immunology and Infection

18.4K 次观看.  Food and Drug Administration. 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.

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

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

酶联免疫斑点法（ELISPOT）:TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

科研

免疫与感染

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

通过流式细胞仪测量附件和流感的内在病毒在A549细胞

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

由成比例的荧光显微镜测量吞噬体pH值

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

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 ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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 ...