作者想感谢夫人莫尼卡Tocchi编辑对稿件的审查。支持AMH是由阿卜杜勒阿齐兹国王大学的奖学金，在加拿大通过沙特阿拉伯文化局。

1。制备试剂和仪器<ol><li>在开始狭缝杂交过程之前，准备的Tris缓冲液（20毫米的Tris，137 mM氯化钠，pH值7.6）公司（TBS）的血凝素，或房委会，狭缝杂交，或0.01％20_mls Zwittergent 4M尿素溶液20_mls在TBS的神经氨酸酶，或NA，狭缝杂交的解决方案。 4M尿素要新鲜，每次准备，10％的DH 2 O Zwittergent原液稳定，在室温下至少6个月，因此可以被摊薄的TBS每次检测前的0.01％。 </li><li>准备2000毫升的洗涤液加入Tween - 20的一个TBS的解决方案，以终浓度为0.1％。脱脂奶粉（印迹级非脱脂奶粉）溶解在洗涤液的终浓度为5％W / V的准备封闭液80毫升。 </li><li>激活的PVDF膜在甲醇15秒（9 × 12厘米大小的预切），其次是2分钟， 在DDH 2 O冲洗浸泡在TBS膜，直到使用。预湿生物点在TBS电视台SF滤纸3张，直到使用。 </li></ol> 2。流感疫苗样品的制备和参考标准房委会的狭缝杂交流感疫苗预先确定相应的被测试的疫苗株的HA含量的参考标准，生物制品评价和研究中心（CBER / FDA，美国）或国家生物标准和控制研究所（NIBSC，英国）的中心，并获得用于狭缝杂交医管局量化。研究人员也可以自己准备的抗原标准，如在参考文献9日和10中所述的既定程序。 <ol><li>对于房委会参考抗原和测试疫苗样品稀释4M尿素/ TBS电视台的“房委会参考抗原股票以下浓度：0，0.0938，0.1875，0.375，0.75，1.5，3和6微克房委会/毫升。拌匀。然后，淡化测试获得疫苗生产商或4M尿素/ TBS电视台在内部用于科研目的的人类使用的疫苗样品，并拌匀。虽然可以运行参考抗原和疫苗样本重复，一式三份是首选。准备每个样品稀释液（相差2倍），使样品的浓度范围内标准曲线。每个参考的抗原或疫苗试验样品稀释运行重复或一式三份，分别准备450μL或650μL。单价A型流感疫苗可以与此狭缝杂交方法进行测试。参考抗原HA含量是以前使用的SRID这是目前使用的流感疫苗制剂的标准测试。 HA含量的SRID确定被用来作为初始参考抗原HA浓度股票为编制狭缝杂交标准曲线的稀释。 </li></ol> 3。 NA狭缝杂交流感疫苗样品和参考标准的准备工作流感疫苗的参考标准，相应的被测试的疫苗株取自生物制品评价和研究中心（CBER / FDA，美国），或为国家生物标准和控制（英国NIBSC，）研究所为中心，为NA量化使用狭缝杂交。这些参考样本NA含量deglycosylated样品经SDS - PAGE分析，结合光密度扫描分析确定，如前所述9，与10个细微的修改。简言之，deglycosylated疫苗的参考样本（5微克）与样品缓冲液混合，并装上凝胶。凝胶运行约90分钟，直到跟踪染料用完的凝胶，SYPRO染色，在20 mA。光密度定量的蛋白质进行了使用氟化凝胶的文件系统（阿尔法INNOTECH公司）。 NA的金额是确定的NA蛋白的比例，用Lowry法测定总蛋白的基础上。使用这种方法制备的参考样本，然后可以使用狭缝杂交疫苗样品中Na的含量从疫苗生产厂家进行量化。 <ol><li> NA参考抗原和测试疫苗样本：NA参考抗原股票Zwittergent / TBS以下浓度：0，0.039，0.078，0.1562，0.3125，0.625，1.25，2.5，5和10μgNA /毫升稀释0.01％。拌匀。然后，淡化测试获得疫苗生产商或0.01％Zwittergent / TBS电视台在内部用于科研目的的人类使用的疫苗样品，并拌匀。虽然可以运行参考抗原和疫苗样本重复，一式三份是首选。准备每个样品稀释液（相差2倍），使样品的浓度范围内标准曲线。每个参考的抗原或疫苗试验样品稀释运行重复或一式三份，分别准备450μL或650μL。单价流感疫苗可以测试机智h本狭缝杂交方法。 </li></ol> 4。 PVDF膜生物点SF微滤设备和样品的杂交大会上<ol><li>组装以前清洁和干燥的生物点SF微滤设备。放入真空歧管垫片支撑板和3蘸生物点SF过滤器在垫片的纸页上top.Place的密封垫片的地方，其次是预先浸泡PVDF膜。将膜和使用对角交叉的方式，在膜表面的压力，以确保统一应用的四个螺丝用手拧紧顶部的示例模板。 </li><li>验证3单向阀设置为只公开之前，转向泵和连接真空的真空歧管的真空源。 </li><li>打开泵和连接点生物仪器。确保以下步骤样品样品的快速和适当的排水井的水平以下的位置，流量阀。 </li><li>重复使用斜穿模式的螺丝拧紧步。拧紧螺丝，而真空应用，确保了更严格的密封，防止井之间的交叉污染。 </li><li>改变流量阀揭露真空歧管，空气和关闭泵。 </li><li>将100μL的TBS使用多通道移液器的所有油井，补充水分膜，从而保证了统一的具有约束力的抗原。必须小心，以避免形成泡沫，并申请在井中间的解决方案，尽可能接近底部，以均​​匀覆盖的插槽。突发无意中用移液器尖推出任何气泡。 </li><li>改变流量阀歧管暴露在空气和真空，打开泵，暴露在空气中的端口，以规管的真空量将超过一个手指轻轻地漏井的缓冲区。 </li><li>改变流量阀歧管暴露在空气中，只要缓冲区已经完全从所有的井排水。关闭泵或断开真空。 </li><li>应用的每个标准或样品稀释疫苗，每孔200μL。一次，在每口井的中心，吸取样品，接近底部尽可能照顾，以避免引入气泡。突发无意中用移液器尖推出任何气泡。将200μL样品稀释缓冲液中未使用的水井（4M尿素/ TBS电视台或0.01％Zwittergent / TBS电视台），以确保适当的真空中使用的水井。 </li><li>调节流量阀，暴露在空气和真空歧管。打开泵（或重新连接真空），并轻轻地使样品完全由一个比暴露在空气中的端口手指控制的真空量从井漏。删除从港口的手指释放真空，只要所有的样品都倒掉。 </li><li>立即加入200μL的TBS到每口井用多通道移液器。应用温和的真空排水和洗井，由一个比暴露在空气中的端口手指控制的真空量。 </li><li>重复步骤4.11所述的清洗步骤。只要井已完全耗尽，样本模板的螺丝松动，关闭真空，以避免overdrying井。立即进行删除膜。 </li><li>膜放入封闭液，孵育过夜在4 ° C，温和的摇摆。使用足够的封闭液完全覆盖膜。 </li></ol> 5。 HA和NA的免疫抗原<ol><li>在封闭液孵育过夜，洗膜5分钟，两次TBS/0.1％Tween - 20的洗涤液。对于所有的洗涤和孵化，确保膜完全覆盖解决方案。 </li><li>孵化膜对HA（如UNI - 1）或NA（如HCA - 2或HCA - 3）的普遍兔抗体稀释于5％W / V BSA洗一小时，在室温下的缓冲区，用温柔摇摆。需要确定每个抗体股票的最佳抗体浓度。兔抗血清1:4000的稀释了上述抗体的最佳结果。 </li><li>洗膜3次，每次15分钟的温和的摇摆在室温下，以消除任何未绑定的主要抗体。 </li><li> HRP标记的抗兔二抗孵育膜。使用1:5稀释的热ImmunoPure山羊抗兔IgG，过氧化物酶标记的抗体阻止缓冲区，并在室温孵育30分钟温和的摇摆。 </li><li>洗膜3次，摇摆每次15分钟，洗涤液在室温下，以消除任何未绑定的二级抗体。 </li><li>膜用TBS洗5分钟温和的摇摆，从膜表面去除剩余吐温20洗涤剂。 </li><li>准备用3毫升每个R基板eagent从Supersignal西杜拉的工期顺延Thermo Scientific和拌匀基板套件。 </li><li>将膜在干燥的纸巾，轻轻地清除多余的TBS缓冲液。 </li><li>膜转移到干燥的容器中，并添加基材，以覆盖整个膜。在室温下孵育5分钟，温和的摇摆。 </li><li>纸巾轻轻印迹膜中删除多余的基板。 </li><li>化学发光膜的膜暴露。曝光的长度，将需要优化，以避免信号饱和，但通常会范围从10秒到10分钟。 </li><li>执行氟化凝胶的文件系统（阿尔法INNOTECH公司）制造商的指示使用凝胶成像装置上发展起来的薄膜的光密度分析。每个参考抗原和测试疫苗样本重复设置的密度值，然后取平均值。虽然可重复运行的抗原标准和疫苗样本，一式三份是首选。为复制的密度值应非常相似。复制之间存在显着差异表明可能在手术过程中的技术问题。浓度依赖性标准曲线的建立与HA或NA抗原的参考标准，在每个插槽的数量（NG）绘制的平均密度值。在免疫中使用适当的校准曲线拟合是HA和NA准确地量化的关键。线性回归，绘制响应（Y）与浓度（X）的响应曲线（Y = MX + B）的线性部分获得的是最简单的方法分析物的定量。如果更广泛的分析物浓度计算认为，目前接受免疫的四个参数物流（4PL）模型应该被使用。各种软件是供有兴趣的研究者考虑。在所描述的狭缝杂交检测情况下，x轴的值转换成数比例允许的曲线，以适应4 - PL模式和使用一个变量的斜坡非线性回归，计算HA和NA的抹杀金额因此，可以测试的疫苗样本。因此，我们经常使用这个模型，我们的分析。无论是4M尿素/ TBS电视台或0.01％Zwittergent / TBS电视台前膜涂抹上，由于测试疫苗样品稀释，每个样品的稀释倍数，需要加以考虑，以确定房委会及Na含量的原始测试疫苗样本。这是建议，每个测试疫苗样本是在为了一套密度值运行在三个不同稀释度（相差2倍），属于标准曲线。密度值比标准曲线范围内的所有测试的疫苗样本稀释高，需要进一步进行准确的HA或NA量化摊薄。如果密度值低于最低的曲线结束，测试样品应在一个较低的稀释倍数稀释。 </li></ol> 6。代表性的成果： 所有可用的A型流感房委会序列的生物信息学分析证实了N -末端的HA2亚基（融合肽）医管局作为唯一的保守区域。图1显示了每个所确定的共识序列的氨基酸位置的保护率。确定了两个变化在位置2（L -&gt;）和12（G -&gt; N）的N -端的14个氨基酸的HA2，但这种差异不影响抗体和肽的6变种之间的绑定。 UNI - 1的抗原表位（GLFGAIAGFIEGGW）被选为发展对医管局的一种普遍的抗体。这种抗体对病毒序列表现出显着的特异性和13不同亚型的A型流感HA（H1 - H13），（图2）结合的能力。 使用类似的方法，两个普遍保守的序列被确定在所有流感一个不适用的，一个比较接近的酶活跃网站（HCA - 2）（图3A）和在Ñ -总站（HCA - 3）其他（图3B） 。这些氨基酸序列的多肽作为抗原，产生对NA普遍抗体。对两个抗原决定簇的抗体能够绑定到9所有的NA亚型，并表现出非常小的交叉反应，尿囊或细胞的蛋白质，从而表现出高特异性病毒NA序列（图4）。 图5显示了流感HA和NA的量化的狭缝杂交方法的概述。 HA和NA抗原槽blot分析的例子，在图6和图7所示。图6A和6C显示后，代表房委会在流感疫苗的参考标准和疫苗样本分别抗原检测结果。运行一式两份，每份样品（标准或疫苗）在相邻井。重复应该显示similař强度以下检测。取决于所用的抗体，抗体稀释液，孵化和曝光时间的优化可能需要。 房委会从疫苗的参考标准样品的不同浓度的检测获得一个典型的标准曲线的一个例子是在图6b所示。狭缝杂交化学发光检测后的HA抗原的浓度是成正比的信号强度，适合这个浓度范围（0.0938至6微克/毫升医管局）4 PL曲线拟合模型。 图7显示了代表流感疫苗的参考标准，并在疫苗样本NA抗原的检测结果。密度分析表明，各种稀释疫苗的参考标准由狭缝杂交NA抗原检测得到的信号强度成正比浓度的NA（图7A）。由此产生的标准曲线拟合一个4 - PL这个浓度范围（0.039至10微克NA /毫升）的模型，如图7b所示。图7C显示流感疫苗样本中的Na含量的狭缝杂交分析的一个例子。一式两份，每份样品检测相邻井。 <img src="/files/ftp_upload/2784/2784fig1.jpg" alt="图1"/> 图1融合肽区域的A型流感房委会UNI - 1的抗原表位的保护率。 生物信息学分析所有可用的A型流感HA序列融合肽（的HA2亚基的N -末端位于）证实，只有普遍保守的地区医管局。请注意，在位置2的变化（L -&gt;我）和12（G -&gt; N）的被发现，不影响抗体结合6。一个普遍的抗体具有约束力的13个不同的亚型的A型流感HA（H1 - H13），能够被开发使用UNI - 1抗原表位（GLFGAIAGFIEGGW）。 <img src="/files/ftp_upload/2784/2784fig2.jpg" alt="图2"/> 图2 13亚型HA对房委会的普遍抗体的检测。 13亚型的A型流感病毒的尿囊液在鸡胚中传播，在SDS - PAGE分割，其次是房委会的使用对房委会的UNI - 1的抗原表位的通用抗体蛋白的检测。 NP蛋白装载控制使用多克隆NP特异性抗体检测。阴性对照组（ - ）是来自未受感染的鸡蛋尿囊液。来自英国NIBSC，阳性对照（+），是H1的参考标准<img src="/files/ftp_upload/2784/2784fig3.jpg" alt="图3"/> 图3。酶的活性部位和N -末端附近的A型流感NA的序列同源性。 两个普遍保守序列被确定在A型流感NA生物信息学的，位于附近的酶的活性部位（HCA - 2）（A组），其他的N -端（HCA - 3）（B组）。这些保守的氨基酸序列的肽合成和用于生成对NA普遍抗体。 <img src="/files/ftp_upload/2784/2784fig4.jpg" alt="图4"/> 图4 9亚型的NA NA抗体的检测。 在鸡胚尿囊九NA A型流感病毒亚型的体液传播，在SDS - PAGE电泳分馏，通过检测的NA使用HCA - 2（A组）和HCA - 3（B组）抗体的蛋白质。 NP蛋白检测为装载控制使用多克隆NP特异性抗体（C组）。阴性对照组（ - ）是来自未受感染的鸡蛋尿囊液。阳性对照（+），尿囊液，添加/新喀里多尼亚RNA1，并与相应的抗血清检测。 <img src="/files/ftp_upload/2784/2784fig5.jpg" alt="图5"/> 图5的HA和NA抗原的流感疫苗的定量狭缝杂交程序流程图。 先准备样品稀释，洗涤和阻塞狭缝杂交膜以及所需的缓冲区和聚偏氟乙烯膜生物点SF微滤设备的组装所需的滤纸预先浸泡。流感疫苗和参考标准样品稀释HA或NA狭缝杂交检测的最佳缓冲区。根据制造商的说明和样本应用到PVDF膜生物点SF微滤设备组装。 HA和NA抗原的检测普遍对每个抗原抗体。 HA和NA的定量获得被测样品的化学发光信号进行光密度分析。 <img src="/files/ftp_upload/2784/2784fig6.jpg" alt="图6"/> 图6。医管局抗原检测在流感疫苗样本，并参考ENCE标准。 A组有代表性的杂交，获得以下的HA抗原的免疫检测。房委会参考抗原稀释到房委会/ ml和疫苗稀释4M尿素/ TBS的HA /毫升0.375微克的浓度，然后被应用到PVDF膜与生物点SF微滤设备从0到6微克的浓度。 HA抗原检测使用UNI - 1抗体普遍。 B组显示疫苗的参考标准，医管局各浓度检测信号得到标准曲线的一个例子。信号强度是成正比的HA浓度和曲线拟合一个4 - PL模式。 <img src="/files/ftp_upload/2784/2784fig7.jpg" alt="图7"/> 图7的流感疫苗样品和参考标准NA抗原的检测。 小组A和B代表信号获得NA的流感疫苗为NA浓度在0.01％Zwittergent / TBS和由此产生的4 - PL以下光密度分析的标准曲线范围从0到10微克NA /毫升稀释的参考标准检测。典型的化学发光法检测到信号后的NA抗原的狭缝杂交分析，在流感疫苗样本的一个例子是在面板C.流感疫苗样品稀释在0.01％Zwittergent / TBS的HA /毫升浓度为1μg，并涂抹上一个PVDF膜使用生物点SF微滤设备。相应的疫苗的参考标准，范围从0到2.5微克NA /毫升的浓度，稀释，以建立一个NA抗原的疫苗样品的定量标准曲线相同的印迹。 NA抗原的检测与HCA - 2抗体普遍对NA和获得是进行化学发光信号的密度分析。可以计算出每种疫苗样本量的Na与NA疫苗的参考标准浓度绘制信号强度值对4 - PL标准曲线。

<ol>
	<li>Webster, R. G., Bean, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+influenza+virus.">Genetics of influenza virus.</a> Annu Rev Genet. 12, 415-431 (1978).</li><li>Skehel, J. J., Wiley, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+binding+and+membrane+fusion+in+virus+entry:+the+influenza+hemagglutinin.">Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.</a> Annu Rev Biochem. 69, 531-569 (2000).</li><li>Wood, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+the+host+cell+on+standardisation+of+influenza+vaccine+potency.">The influence of the host cell on standardisation of influenza vaccine potency.</a> Dev Biol Stand. 98, 183-188 (1999).</li><li>Sylte, M. J., Suarez, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+neuraminidase+as+a+vaccine+antigen.">Influenza neuraminidase as a vaccine antigen.</a> Curr Top Microbiol Immunol. 333, 227-241 (2009).</li><li>Bright, R. A., Neuzil, K. M., Pervikov, Y., Palkonyay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+meeting+on+the+role+of+neuraminidase+in+inducing+protective+immunity+against+influenza+infection.">WHO meeting on the role of neuraminidase in inducing protective immunity against influenza infection.</a> Vaccine. 27, 6366-6369 (2008).</li><li>Chun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+antibodies+and+their+applications+to+the+quantitative+determination+of+virtually+all+subtypes+of+the+influenza+A+viral+hemagglutinins.">Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins.</a> Vaccine. 26, 6068-6076 (2008).</li><li>Gravel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+analyses+of+virtually+all+subtypes+of+influenza+A+and+B+viral+neuraminidases+using+antibodies+targeting+the+universally+conserved+sequences.">Qualitative and quantitative analyses of virtually all subtypes of influenza A and B viral neuraminidases using antibodies targeting the universally conserved sequences.</a> Vaccine. 28, 5774-5784 (2010).</li><li>Li, 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+simple+slot+blot+for+the+detection+of+virtually+all+subtypes+of+the+influenza+A+viral+hemagglutinins+using+universal+antibodies+targeting+the+fusion+peptide.">A simple slot blot for the detection of virtually all subtypes of the influenza A viral hemagglutinins using universal antibodies targeting the fusion peptide.</a> Nat Protoc. 5, 14-19 (2010).</li><li>Harvey, R., Wheeler, J. X., Wallis, C. L., Robertson, J. S., Engelhardt, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+haemagglutinin+in+H5N1+influenza+viruses+reveals+low+haemagglutinin+content+of+vaccine+virus+NIBRG-14+(H5N1).">Quantitation of haemagglutinin in H5N1 influenza viruses reveals low haemagglutinin content of vaccine virus NIBRG-14 (H5N1).</a> Vaccine. 26, 6550-6554 (2008).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+deglycosylation+and+electrophoresis+to+the+quantification+of+influenza+viral+hemagglutinins+facilitating+the+production+of+2009+pandemic+influenza+(H1N1)+vaccines+at+multiple+manufacturing+sites+in+China.">Application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of 2009 pandemic influenza (H1N1) vaccines at multiple manufacturing sites in China.</a> Biologicals. 38, 284-289 (2010).</li><li>Johansson, B. E., Pokorny, B. A., Tiso, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supplementation+of+conventional+trivalent+influenza+vaccine+with+purified+viral+N1+and+N2+neuraminidases+induces+a+balanced+immune+response+without+antigenic+competition.">Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition.</a> Vaccine. 20, 1670-1674 (2002).</li><li>Hashem, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+antibodies+against+the+highly+conserved+influenza+fusion+peptide+cross-neutralize+several+subtypes+of+influenza+A+virus.">Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus.</a> Biochem Biophys Res Comm. , (2010).</li></ol>

流感病毒的HA和NA的定量测定疫苗的研究和开发的关键，因为这两个表面蛋白是最重要的诱导免疫反应6-11的病毒成分。此前报道的免疫学方法对这些蛋白质的检测需要应变的特异性抗体。简单，重复性好，快速的狭缝杂交的方法来量化这里描述的HA和NA抗原是适用于所有A型流感病毒的HA和NA蛋白抗体识别只有普遍保守的抗原决定簇 6-8 。在这个方法感兴趣的调查的唯一的困难可能是?...

An author's affiliation was omitted from the publication of <a href="http://www.jove.com/video/2784">Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays</a>.

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

<table border="1"><tbody><tr><th>试剂或设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>生物点SF微滤器械</td><td> Bio - Rad公司</td><td> 170-6542 </td><td></td></tr><tr><td>生物点SF过滤纸</td><td> Bio - Rad公司</td><td> 162-0161 </td><td></td></tr><tr><td>的Immobilon - FL转印膜（聚偏氟乙烯） </td><td> Millipore公司</td><td> IPFL00010 </td><td></td></tr><tr><td>真空泵</td><td> Millipore公司</td><td> WP6111560 </td><td></td></tr><tr><td>化学发光百玛士光膜</td><td>柯达</td><td> 178 8207 </td><td></td></tr><tr><td>氟化凝胶文件系统</td><td>阿尔法INNOTECH公司</td><td> 29 - 008 - 1896X </td><td></td></tr><tr><td>针对HA和NA抗原的通用兔抗体</td><td></td><td> UNI - 1（“房委会”）HCA - 2，HCA - 3（NA） </td><td>抗体可通过MTA或有兴趣的研究者可以根据先前所描述的6,7的程序产生。 </td></tr><tr><td>流感疫苗参考抗原</td><td> CBER / FDA或NIBSC </td><td></td><td></td></tr><tr><td>流感疫苗样本</td><td></td><td></td><td>在大多数国家普遍使用</td></tr><tr><td>尿素</td><td> Sigma - Aldrich公司</td><td> U1250 </td><td></td></tr><tr><td> Zwittergent 3-14洗涤剂</td><td> Calbiochem </td><td> 693017 </td><td></td></tr><tr><td>吐温20 </td><td> Fisher Scientific则</td><td> BP337 - 500 </td><td></td></tr><tr><td>杂交级拦截器非脱脂奶粉</td><td> Bio - Rad公司</td><td> 170-6404 </td><td></td></tr><tr><td> ImmunoPure山羊抗兔IgG（H + L），过氧化物酶</td><td> Thermo Scientific的</td><td> 31460 </td><td></td></tr><tr><td> SuperSignal西DURA工期顺延基板</td><td> Thermo Scientific的</td><td> 34075 </td><td></td></tr></tbody></table>

quantitative analyses of all influenza type a viral hemagglutinins and neuraminidases using universal antibodies in simple slot blot assays

血凝素（HA）和神经氨酸酶（NA）是流感病毒，这是众所周知的发挥重要作用，在病毒的生命周期和诱导免疫反应的保护1,2两个表面蛋白。由于中和抗体的主要目标，房委会是目前使用的流感疫苗的效力标记和测量单向免疫扩散法（SRID）3 。然而，提供相应的亚型特异性血清的SRID的依赖，导致延迟2-3个月的最低，为每一个新的疫苗版本。此外，尽管有证据表明，娜也诱导保护性免疫，NA的流感疫苗数量，由于缺乏适当的试剂或分析方法5尚不规范。因此，简单的量化HA和NA抗原的替代方法是可取的快速释放和更好的流感疫苗的质量控制。 在所有可用的A型流感HA和NA序列普遍保守区域确定了生物信息学分析6-7。只有在普遍保守的HA，抗原表位的融合肽的6的，而两个保守序列neuraminidases，一靠近酶的活性部位（HCA - 2）和一个序列（UNI - 1）被确定其他接近的N -端（HCA - 3）7。这些氨基酸序列的肽合成和用于免疫家兔抗体的产生。对房委会的UNI - 1的抗原表位的抗体能够绑定到13亚型的A型流感HA（H1 - H13），而对HCA - 2和HCA - 3地区的NA的抗体能够有约束力的全部9个NA亚型。所有的抗体对检测尿囊蛋白无交叉反应的观察证明，病毒序列显示了显着的特异性。这些普遍的抗体，然后用于发展狭缝杂交分析，量化而不需要特定抗血清 7,8的HA和NA在A型流感疫苗。疫苗样本应用到PVDF膜的使用以及与不同浓度稀释到的参考标准插槽印迹装置。医管局检测，样品和标准，先稀释在Tris缓冲液（TBS）含有4M尿素，而NA的测量他们的TBS稀释含有0.01％Zwittergent这些条件显着提高了检测的灵敏度。按照各自的普遍抗体免疫印迹的HA和NA抗原的检测，密度测量信号强度进行了量化。 HA和NA在疫苗的数额，然后使用标准曲线的建立与使用引用不同浓度的信号强度计算。 鉴于这些抗体结合在HA或NA普遍抗原表位，有兴趣的研究者可以作为研究工具使用，他们在其他免疫仅比狭缝杂交。

Watch this Scientific Journal Video about Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays at JoVE.com

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

一个简单的狭缝杂交方法被开发用于流感病毒的血凝素和使用针对其最保守的序列，通过生物信息学分析确定的普遍抗体神经氨酸酶的量化。这种创新的方法可以提供一个有用的替代所有病毒的血凝素和神经氨酸酶的定量测定。

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 ...

quantitative-analyses-all-influenza-type-viral-hemagglutinins

Research

JoVE Journal

Immunology and Infection

18.1K 次观看.  Health canada. 一个简单的狭缝杂交方法被开发用于流感病毒的血凝素和使用针对其最保守的序列，通过生物信息学分析确定的普遍抗体神经氨酸酶的量化。这种创新的方法可以提供一个有用的替代所有病毒的血凝素和神经氨酸酶的定量测定。

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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

牛痘病毒记者量化病毒功能在基因表达的各个阶段

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

病毒浓度的测定通过空斑试验：用传统的和新型覆盖系统

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

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 ...

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

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 ...