这项工作由斯克里普斯芯片核心设施，并从疾病控制中心（JCP）合同的一部分得到了支持。 RPdV是来自荷兰科学研究组织（NWO）一个人Rubicon和VENI授权的收件人。由NIGMS授予GM62116（JCP）资助的财团为功能糖组学（http://www.functionalglycomics.org/）提供在这项研究中使用的几个聚糖。这是来自斯克里普斯研究所的出版物29113。

注:所有步骤都在室温下进行，除非另有说明。 1.阵列建设<ol><li>多糖制备及板设置<ol><li>准备打印缓冲区的股票。首先使500毫升磷酸氢钠的150mM的储备液。也使50毫升磷酸二氢钠溶液的150毫原液。在烧瓶中，使用磁力搅拌棒和pH计，直到达到pH为8.5缓慢滴定与一元溶液的磷酸氢钠溶液中。添加吐温-20〜0.005％。 </li><li>准备聚糖样品。稀释PAA结合聚糖（diLacNAc（Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA），3SLNLN（Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA）和6SLNLN（Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA（ 图1A ））被以稀释至100μG/ ml，在从step1.1.1。单价聚糖打印缓冲器，diLacNAc（Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-（CH 2）3 NH 2，3SLNLN（Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ - （CH 2）3 NH 2）和6SLNLN（Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1- 4GlcNAcβ - （CH 2）3 NH 2）），以100μM在从步骤1.1.1打印缓冲器。最后，准备胺官能染料，如方格标记/着陆灯使用。染料标记点是在1μM打印。 注:Atto488-NHS染料中的打印使用。然而，任何胺官能染料，由滑动扫描器可读适合此目的。 </li><li>转移10微升每聚糖样品至384孔微量滴定板的，用于印刷的机器人。将未使用的聚糖在在密封管-20°C的打印缓冲溶液。 Tansfer 10微升染料标记印刷板。 </li></ol></li><li>印花 注意:所有STEps的这种担心触摸或移动必须用手套来完成的幻灯片。 <ol><li>根据布局的正确配置放置样仪销在打印头。此布局使用一个针打印所有样品和阵列。打印聚糖六块，跳过一个特征（特征被定义为某种化合物的单点），在每一侧，以使得相同样品的斑点不完全打印彼此旁边。 注:最终用户必须决定的配置。 </li><li>从袋，打开箱子除去幻灯片，并除去任何灰尘颗粒或小片的塑料，可能通过在每个滑动吹超高纯度氮气是在其表面上。放置幻灯片的期望数量，涂布面朝上，在点样仪阶段。 </li><li>节目的点样仪，使用的每次访问27点参数的源板，用3"预点"到聚-L-赖氨酸涂覆的载玻片，在打印针的尖端，以除去过量的液体<ol><li>打开MPU（主电源单元）和气候控制单元。打开计算机上平铺分析软件。 </li><li>选择布局打印。选择要用于打印阵列1×1的打印工具选项→针工具 - 1单针。选择目标选项卡→工具数组定义→打印模式与添加的间距（点到点的距离），将要打印的行和容纳样品的列的数量，以及样品的印刷图案（此阵列的8 ×8印刷图案在340微米间距为7总样本）中使用。 </li><li>选择目标→幻灯片版式和编程块到块的距离，让每个重复阵列打印到48孔（4×12）幻灯片模板。选择源和（当使用1针1对每个样品）（此打印7来源皮卡将被用来容纳6样品孔和1个染料标记井）添加源皮卡的数量。 注:源对话应该把绿色，以显示该号码源的访问匹配的印刷图案要打印的样品。 </li><li>选择目标→适配器板和幻灯片布局和调整幻灯片编号（5张幻灯片都是一次打印此打印1预去斑幻灯片）。 </li><li>通过点击T1按钮来激活托盘和添加幻灯片的所需数量的托盘，密切关注点样前滑（蓝色）和"真实的"幻灯片的位置释放托盘。 </li><li>广场上所有剩余的真空端口平原载玻片阻止真空，然后单击确定以激活真空。检查所有幻灯片就位，然后单击确定托盘返回初始位置。 </li><li>装载印版到所述板夹持器，并确保齿条来代替设置正确。点击GO，让打印过程继续进行。 </li></ol></li><li>打印中，每个阵列6点重复其余24个点（4阵列/源的访问）。 </li><li>加载384孔板装入打印机，开始打印。保持相对湿度，在打印时，在整个打印之间55和65％。打印5张幻灯片后，打印机停止。 注意:虽然在此测定法与微阵列中的特定的8×8图案被印刷，不同的微阵列印刷机器人将需要设备特定的编程来实现所需的布局。它是由最终用户来确定给定的其设备的规格的最适当的模式。 </li></ol></li><li>加湿/固定化 注意:一旦幻灯片打印结束后，他们接受一个临时的固定化步骤，也称为加湿。加湿印刷后有助于均匀通过重新润湿斑点，并确保完全阻断样品附件。 <ol><li>放于100％湿度室玻片一段30分钟印刷后立即。通过将非常湿纸巾平在一个大的玻璃盘的底部，放置幻灯片构造腔室关于某种架上，诸如试管架，打印面朝上的，并用保鲜膜套住中的水分密封。 </li><li>数用酒精/耐溶剂标志着一个干燥盒笔和存储幻灯片。变干过夜。 </li></ol></li><li>编号，阻塞和存储<ol><li>准备封闭液 - 50毫米乙醇胺在50 mM的硼酸盐缓冲液。首先，溶解硼酸，在DDH 2 O至50mM的终浓度在含有磁搅拌棒的烧瓶中。大力搅拌溶液在搅拌盘，直到所有的固体溶解。同时不断搅拌，从16.54 1M贮存液中添加乙醇胺适量以达到预期的50mM的浓度。添加浓氢氧化钠调整到9.2的最终pH。 </li><li>浸入印刷载玻片在1小时的封闭缓冲液溶液。 </li><li>继1小时，冲洗的幻灯片在DDH 2 O，除去封闭缓冲任何多余的痕迹。配置适当的废物容器封闭液。 </li><li>转移幻灯片玻璃染色持有者和甩干。干阵列通过将它们放置在配备有摆动板持有者离心机，在10 xg的转速为5分钟。 </li><li>一旦载玻片是干的，它们可以立即孵育或存储。储存条件是在一个密封的塑料袋-20℃。 </li></ol></li></ol> 2.分析聚糖结合蛋白<ol><li>制备加湿室，其中待杂交的阵列将适合。一个简单的室由耐热盘，在分层蘸用纸巾和一个机架，在其上的幻灯片将休息底部。 </li><li>使用生物素化凝集素的SNA（ 桑布卡黑凝集素）和ECA（ 刺桐cristagalli凝集素）以10微克/毫升+ 2微克/毫升链霉-555染料，在探测缓冲液（PBS + 0.01％吐温-20）。对于HA（A /越南/ 1203年至1204年的H5N1和A / KY / 07 H1N1，在这个例子中），使用20微克的起始浓度/ ml的预络合，如前所述34。简要地说，使HA:鼠标-α-链球菌:山羊-α-鼠标Alexa647混合物在封闭缓冲液（PBS + 3％BSA + 0.01％吐温-20）中，在4:2:1的摩尔比。 注:该阵列探测与外源凝集素以确定聚糖被固定和可检测（ 图1B）。 </li><li>的20微升聚糖结合蛋白 - 抗体混合溶液转移至384孔板中并在冰上孵育30分钟。连续稀释凝集素和HA样品1:1，在其合适的缓冲液，8倍，通过将10微升的样品与10微升缓冲液中。 </li><li>吸管8微升样品到微孔表面和孵育在90分钟密封加湿（100％RH）下室中。 </li><li>通过保持边缘和浸渍他们四次和0.05％吐温的PBS的混合物洗载玻片一次一个，然后四PBS倍，并在去离子H 2 O最后四次运用在封闭步骤中描述的相同的干燥协议，旋阵列干燥在离心机中在10×g下5分钟。 </li></ol> 3.扫描和数据分析 注:聚糖微阵列可以在不同的设置，扫描取决于芯片扫描仪制造商。通过改变激光功率和分辨率，该仪器可以产生与它的动态范围（ 图1C）内尽可能多的信号尽可能的图像。 <ol><li>使用荧光滑动扫描器，并与聚糖结合蛋白孵育后，立即扫描阵列。注意确保幻灯片正确插入扫描仪。 <ol><li>开扫描软件。单击开始以打开该程序。连接到扫描仪:扫描仪→连接或绿色按钮在扫描仪的导航面板菜单。 </li><li>扫描仪打开门自动加载磁带机。放入插槽幻灯片在特定的顺序自动加载机。关闭自动加载磁带机门。氯ICK"读装载机"中的工具导航面板自动检测扫描仪的幻灯片。 </li><li>设置扫描参数:合适的实验（ 如 635激光功率高，检测增益50％）扫描仪→扫描参数。扫描幻灯片:扫描仪→扫描。通过创建一个文件夹来保存扫描图像和名称单独扫描选择路径。做到这一点之前，实际的扫描。重复每个单独的幻灯片。单击确定开始扫描。 </li></ol></li><li>使用扫描仪软件来设置仪器和扫描的幻灯片。设置扫描器迭代扫描在低激光功率（5毫瓦）随的25％，50％和75％的增益设置。 注:在设置中进行测试和优化，但要记住，每次扫描可降低可能由于漂白染料的荧光输出。 <ol><li>开放数据采集和分析软件如MAPIX软件。单击开始以打开该程序。打开扫描图像:文件→打开聊天室年龄。打开GAL文件:分析→开放式网格并选择适当的GAL文件。 </li><li>进入块模式:图像→模式的块移动的网格。使用网格标记到网格设置在滑动的适当位置。调整各个块所必需的印刷阵列相匹配。 </li><li>进入点模式:图像→点模式来调整位置和块内的个别景点的大小。一旦所有的块和斑点调整到位，措施的信号强度通过分析→光度计算。减输出文件。 </li></ol></li><li>一旦载玻片完成扫描，图像分析用于与安装的（ 图 1D - G）的图像处理程序的结合的信号。对于图像分析，加载在扫描TIFF图像一个GAL（数组列表）文件。 注意:GAL文件包含当场布局模式，每个点的位置的标识。数组GAL文件是CR通过使用包含样品的身份和他们在384孔源板位置的制表符分隔文本文件中的打印机软件eated。 </li><li>使用印在芯片格标记/着陆灯要正确放置GAL网格。个别位置可能需要被单独移动，但一个印刷井阵列通常将允许在网格内的块放置自如。下面的网格布局，由软件收集点强度，并保存为制表符分隔文本文件（.txt）。 <ol><li>使用电子表格程序，打开扫描仪​​的输出文件。从打开的文件夹位置适当的宏文件。点击查看→宏→RunMacro。 注意:预写将复制的原始输出数据，删除不必要的行和列，由试样的数据进行排序，计算平均平均信号减去背景值，并在绘制所得值以包含每个图表的电子数据表的工作表六SAMP的莱斯阵列上进行测试。 </li></ol></li></ol>

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The authors have nothing to disclose

评估IAV的受体特异性在分析禽流感病毒大流行潜力的重要一步。由病毒唾液酸识别被链接到几个生物性能如从细胞结合和释放。其中知识氨基酸的突变是必要的禽流感病毒实现α2-6结合和跨越物种屏障使大流行的准备。数测定法用于确定受体特异性;但是，所有有其缺点，包括尺寸仅为亲和力，而不是特异性， 反之亦然 。 这里，我们描述基于广泛使用和接受的ELISA，一?...

野生水禽是IAV的自然宿主，但是IAV能够跨越物种屏障，禽类和哺乳动物，包括人类。禽流IAVs认识α2-3连接的唾液酸（禽型受体），而人类病毒结合α2-6连接的唾液酸（人型受体）。为了能够有效复制和人类禽类IAV需要绑定到人型受体1之间传递。 IAVs是基于血清学表征其血凝素（HA）和神经氨酸酶（NA）包膜糖蛋白的抗原性划分。 HA结合到唾液酸，而NA是在病毒的生命周期并切割唾液酸2的另一端的受体破坏酶。所有的人感染病毒，包括H1N1，H2N2和H3N2，有禽源3。在过去的二十年中几个禽类到人类交叉的发生，用H5N1，H7N7，和H7N9是最公知的;豪版本，其他亚型传染人类更零星（H6N1，H7N1，H7N2，H9N2，H10N7，H10N8）4。幸运的是，似乎没有这些病毒已经能够完全适应人型α2-6连接的唾液酸受体5-8。禽流感或其他人畜共患的病毒适应，以适应人类宿主复制和传播可能对人体健康产生破坏性的影响。因此，这些病毒是如何发展结合人型受体将有助于新兴流感病毒全球范围内监视的先验知识。 受体偏好的测定采用不同物种的红细胞首次阐明并保持流感研究者9-12之间的青睐检测。即禽流感病毒识别α2-3唾液酸α2-6连接的唾液酸人类病毒的示威最初是基于使用酶设计包含每个李红细胞血凝化验nkages 13,14。虽然读出是血细胞凝集，一个标准测定为病毒学家，底层聚糖结构没有定义，只有终端联系​​。此外，唾液酸转移酶，用于重新sialylate细胞的有限的可用性，已经限制了使用该测定15-18中。接着，使用连接于聚丙烯酰胺（PAA）或聚谷氨酸（PGA）的结构在基于板的测定法19,20唾液酸化聚糖结构中引入确定受体结合偏好的其他方法。几种变化是在涂覆或聚糖或病毒微量滴定板，其中每一个导致一个健壮的，可靠的和非常敏感的ELISA型测定21-23可能。可替代地，生物素连接的聚糖可以取代的PAA / PGA和可以缀合到链霉抗涂层板2,24。虽然可能需要一些特定的血清，酶联免疫吸附都与PAA标准和几个聚糖容易commercially和非商用（财团为功能糖组学（http://www.functionalglycomics.org））。 聚糖微阵列技术已成为一个宝贵的工具，以确定受体特异性，为多个不同的聚糖发现，并结合到多种不同结构可以在单一实验中25-29评估。 IAV的这些结构的结合提供了更好的理解，即IAV优先识别30-33的聚糖结构。聚糖微阵列需要少量的样本量来进行结合分析，只有使用每点（2 NL）聚糖微量。然而，这些阵列通常仅用于评估聚糖受体特异性。的多种病毒，或血凝素蛋白，在多个浓度范围分析可以望而却步由于所需的幻灯片的数量。此外，迄今为止，没有相对亲合力测定已使用聚糖芳开发射线技术。 结合由聚糖微阵列技术和基于ELISA的测定法的PAA联聚糖的灵敏度，得到低样品要求，我们试图开发一个多井聚糖阵列将允许对具有相似或更好的分辨率高通量分析相比传统的基于ELISA的测定法。同时，我们希望最小化消耗的生物制品和报道的化学品的量。最终的结果是一个小型化的亲合力测定中，专门用于监测IAV特异性开发的，同样适用于评估其他聚糖结合蛋白。使用由聚四氟乙烯面具分为48微孔载玻片，6种不同的聚糖看准每孔6个重复。该芯片平台能提供受体趋势相同绑定看到在宏观ELISA格式几个优点。这些包括（I）中6次重复化合物的印刷，使用最少的样品，与若干行的涂布我呐板，使用每孔100μl; （二）多种不同的化合物在单井，包括控制同时进行分析; （Ⅲ）在培养体积和大规模减少; （IV）使用荧光读出一个较大的动态范围。单个幻灯片可以被计算为等于18倍的96孔板。 用下面的协议，任何实验室能够制造和分析的斑点的微阵列应该能够制造这种小型化的ELISA形式。

<table>
	<tbody>
		<tr>
			<td>NEXTERION&#174; Slide H MPX-48&#160;</td>
			<td>Schott</td>
			<td>1091525</td>
			<td>Microwell slides</td>
		</tr>
		<tr>
			<td>ProScanArray Plus&#160;</td>
			<td>PerkinElmer</td>
			<td>discontinued</td>
			<td>confocal microarray scanner</td>
		</tr>
		<tr>
			<td>Innoscan 1100AL Scanner/Mapix Software</td>
			<td>Innopsys</td>
			<td>-</td>
			<td>confocal microarray scanner</td>
		</tr>
		<tr>
			<td>MicroGrid II&#160;</td>
			<td>Digilab</td>
			<td>-</td>
			<td>microaray printer</td>
		</tr>
		<tr>
			<td>SNA</td>
			<td>Vector Labs</td>
			<td>B-1305&#160;</td>
			<td>Plant Lectin</td>
		</tr>
		<tr>
			<td>ECA</td>
			<td>Vector Labs</td>
			<td>B-1145&#160;</td>
			<td>Plant Lectin</td>
		</tr>
		<tr>
			<td>Anti-Strep Antibody</td>
			<td>IBA</td>
			<td>2-1509-001&#160;</td>
			<td>Anitbody for HA binding</td>
		</tr>
		<tr>
			<td>Anti-Mouse Alexa-647</td>
			<td>Life</td>
			<td>A-21235</td>
			<td>Anitbody for HA binding</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287&#160;</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>di-basic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>255793</td>
			<td>printing buffer component</td>
		</tr>
		<tr>
			<td>mono-basic Sodium phosphate</td>
			<td>Sigma</td>
			<td>229903&#160;</td>
			<td>printing buffer component</td>
		</tr>
		<tr>
			<td>poly-l-lysine solution</td>
			<td>Sigma</td>
			<td>P8920&#160;</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma</td>
			<td>221465</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma</td>
			<td>493546</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>incubation/washing buffer</td>
		</tr>
		<tr>
			<td>SMP4B pins</td>
			<td>Telechem</td>
			<td>SMP4B</td>
			<td>printing pin</td>
		</tr>
		<tr>
			<td>Compressed Nitrogen (Grade5)</td>
			<td>Praxair</td>
			<td>UN1066</td>
			<td>general dusting/drying tool</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma</td>
			<td>B6768-500G</td>
			<td>Slide blocking reagent</td>
		</tr>
		<tr>
			<td>ethanolamine</td>
			<td>Sigma</td>
			<td>E9508-500ML</td>
			<td>Slide blocking reagent</td>
		</tr>
		<tr>
			<td>Atto 488</td>
			<td>AttoTec</td>
			<td>AD 488-91</td>
			<td>Gridmarker on array</td>
		</tr>
		<tr>
			<td>PAA-LNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA368</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>PAA-3SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA362</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>PAA-6SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA343</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>LNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te98</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>3SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te175</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>6SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te176</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>384-well microtiter plate</td>
			<td>Matrix TechCorp</td>
			<td>4361</td>
			<td>Printing plate</td>
		</tr>
		<tr>
			<td>VWR lab marker</td>
			<td>VWR</td>
			<td>52877-150</td>
			<td>Slide Numbering</td>
		</tr>
		<tr>
			<td>Wheaton slide staining dish</td>
			<td>Sigma</td>
			<td>Z103969-1EA</td>
			<td>Blocking and Drying</td>
		</tr>
	</tbody>
</table>

a miniaturized glycan microarray assay for assessing avidity and specificity of influenza a virus hemagglutinins

A型流感病毒（IAV）血细胞凝集素识别在细胞表面上的唾液酸作为功能​​性受体来获得进入细胞。野生水禽是IAV的自然宿主，但是可以IAV跨越物种屏障家禽，猪，马和人。禽流感病毒识别由α2-3键（禽型受体）附连到倒数第二个半乳糖，而人类病毒优先识别唾液酸与α2-6键（人型受体）唾液酸。并监控如果禽流感病毒正在适应人型受体，可使用几种方法。用合成唾液酸糖苷的多样库聚糖微阵列越来越多地用于评估受体特异性。然而，这种技术不用于测量亲合力。亲合力的测量一般是通过评估系列稀释血凝素或病毒的结合以吸附到常规聚丙烯96孔板聚糖实现。在该测定中，聚糖与α2-3或α2-6唾液酸连接到生物素并吸附到链霉板，或连接到聚丙烯酰胺（PAA），其直接吸附到塑料。我们通过直接印刷的PAA联唾液酸糖苷及其非PAA联的对应于微井载玻片已显著小型化该测定。这种设置，与在单个幻灯片48阵列，使得能够6聚糖8稀释结合蛋白，询问6个不同的聚糖，包括两个非唾液酸化的控制的同时测定。这相当于在传统的平板测定18倍的96孔板。该聚糖阵列格式降低化合物和生物制品的消费，从而大大提高了工作效率。

打印，扫描和数据分析为确保正确打印，至关重要的是有聚四氟乙烯面罩内的斑点网格，它描绘了在幻灯片上的每个阵列的正确对准。在印刷中，由于聚四氟乙烯涂层的性质，点不能由上MPX幻灯片肉眼看到。注意的是然后在聚-L-赖氨酸包被的玻片预斑点的外观。印刷后直接，各滑动应当由眼对每个斑点化合物是可见的存在，由...

Watch this Scientific Journal Video about A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins at JoVE.com

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

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 ...

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Research

JoVE Journal

Immunology and Infection

7.9K 次观看.  The Scripps Research Institute. 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.

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

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 ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

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

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

ampliPHOX比色法检测为流感的DNA芯片

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

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, ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

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 ...

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.

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

摘要

探索更多视频

此视频中的章节

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

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

ampliPHOX比色法检测为流感的DNA芯片

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

流感病毒的高通量检测方法

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

鼻湿巾从猪流感病毒检测和隔离

在甲型流感病毒的衣壳的体外拆卸梯度离心

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

流感病毒在小鼠感染模型中的研究

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

摘要

探索更多视频

此视频中的章节

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

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

ampliPHOX比色法检测为流感的DNA芯片

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

流感病毒的高通量检测方法

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

鼻湿巾从猪流感病毒检测和​​隔离

在甲型流感病毒的衣壳的体外拆卸梯度离心

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

流感病毒在小鼠感染模型中的研究

鼻湿巾从猪流感病毒检测和隔离