This work was supported by the Academy of Finland (130750 and 218080 to J.T.H.), the Wellcome Trust (090532/Z/09/Z; 089026/Z/09/Z to T.A.B.), and by the MRC (MR/J007897/1 to J.T.H and T.A.B; MR/L009528/1 to T.A.B.).

为计算路线和病毒糖蛋白的尖峰随后均得到详细的协议概述。协议如下图1中所示的工作流程，并结合一种自动化搜索使用初始模板结构和黄金标准结构细化的尖峰。 输入数据，该协议是一组病毒体的断层重建的。一个断层图像中包含的一个或多个病毒粒子。最初，尖峰的一小部分是手工采摘并用来平均水平，完善两个独立的模型。这些模型用于自动定位尖峰上的所有病毒颗粒。最后，两个独立的精炼进行运行，将所得的平均值进行比较，并结合以产生最终的结构。 细化方法证明了使用程序从Jsubtomo包。从Bsoft包28程序用于一般图像Processing任务和分子图形包UCSF嵌合体29是用于可视化的结果。个别课程以斜体字和文件格式，给出的名称标注有大写的文件扩展名。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig1highres.jpg" width="600" /> 图1：用于确定从形性膜病毒糖蛋白刺突复合物的结构总体战略中的数字对应的协议在不同的章节<a href="https://www.jove.com/files/ftp_upload/51714/51714fig1highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 1，从全尺寸断层提取病毒分卷<ol><li>在X线断层手动拾取的病毒颗粒。 <ol><li>打开bshow断层图像文件。定义一个病毒partic的中心坐标使用乐颗粒采摘工具。重复此步骤，直到所有的病毒粒子都被处理。保存病毒坐标在星形文件。 </li><li>直到所有的断层图像进行了处理，重复步骤1.1.1。请记下病毒粒子直径的像素。 注：如果尖峰很难看到断层，使用bfilter到低通滤波器的断层图像，以80的分辨率（参数“-bandpass 80,10000”）。 </li></ol></li><li>运行jsubtomo.py中提取模式（参数“--mode提取物”）中提取病毒分卷到单个卷中的文件。使用保存在步骤1.1.1输入文件STAR文件。 <ol><li>给一大小（参数“--size”）〜比在数据集的最大病毒体大25％。 例如 ，如果病毒是〜200个像素的直径，使用尺寸250×250×250像素。 </li><li>病毒粒子体积的输出文件将在MAP格式。此外，创建一个伴随STAR文件为每个病毒粒子的体积（parame之三“--output”）。 </li></ol></li><li>正常化病毒卷bimg（参数“-rescale 0,1”）。 </li></ol> 2，生成两个独立的初始模型<ol><li>匹克在病毒分卷尖峰的一个子集。 <ol><li>打开bshow病毒体分卷映射文件。定义使用粒子采摘工具，通过工具箱窗口访问穗的中心坐标。重复此步骤，直到所有分明尖峰进行了处理。保存秒杀坐标在星文件。 </li><li>如有必要，重复步骤2.1.1其他病毒体，直到大约200糖蛋白尖刺已被处理。注意像素秒杀尺寸。 注：〜200尖峰是一个粗略的指引，更可能需要的一些应用。如果可能的话，目的是挑选尖峰在不同的方位。避免只采摘俯视图。 </li></ol></li><li>通过运行jviews.p分配一个初始视图向量的尖峰年。使用步骤2.1.1输入文件生成的STAR文件。 注：此图矢量近似于参照病毒的刺突的方向。 <ol><li>使用中央协调分配意见的球形病毒颗粒。例如，如果病毒体被集中（步骤1.2之后）在一个盒子具有250×250×250个像素的大小，中心坐标是125125125像素（选项“--Radial 125125125”）。 </li><li>要指定一个丝状病毒的看法，通过使用在步骤2.1.1作为输入文件中定义的文件之星打开bshow每个病毒分卷。限定使用灯丝拾取工具灯丝的两端点，并保存STAR文件。重复此步骤，直到所有明星的文件已被更新并运行jviews.py使用更新之星文件作为输入文件（--option段）。 </li></ol></li><li>使用jsubtomo_create_mask.py产生真正的空间屏蔽。确保在实际空间掩模是同一三维地球的离子作为将被用于平均的结构（参见2.6.2），并足够大以包含两个尖峰和底层膜的贴剂。 </li><li>使用jsubtomo_create_wedgemask.py产生倒易空间的面具。倒数空间掩码用于排除的区域中的“缺失槽楔&#39;从单一轴断层数据收集所得的区域。确保它是相同的尺寸，为将用于所述平均结构（见2.6.2）。 </li><li>生成一个选择文件（SEL文件）限定属于设定“1”和“2”使用jsubtomo_evenodd.py并在步骤2.2中产生的STAR文件的病毒粒子。 </li><li>使用jsubtomo_create_averages.py产生两个初始平均值。 <ol><li>使用步骤2.5作为输入文件生成的SEL文件。 </li><li>举一个大小（参数“--size”）至少32像素比秒杀的最大尺寸。 例如 ，如果秒杀〜40个像素长，使用尺寸72×72×72像素。 </li><li>适用于高对称性（ 如 C100）的平均值（参数“--symmetry C100”），近似围绕秒杀的长轴圆柱形的平均水平。使用对称，以减少在初始平均噪声。 </li><li>项目（参数“--suffix”）的输出文件提供一个唯一的名称。 </li><li>使用在步骤2.3中产生的真实空间掩模掩盖远背景（参数“--mask”）。使用在步骤2.4中产生的倒易空间掩模占信号由丢失楔（参数“--mask”）的存在下引入的损耗。 </li></ol></li></ol> 3，黄金标准迭代对齐和两个初始穗模型的平均反复调整和平均在第2与jsubtomo_iterate_gold.py生成的两个初始模型。 <ol><li>阶段一，这个阶段的目的是改进观察向量的方向。输入文件是在步骤2.5生成的SEL文件。 <ol><li>使用一个8度角取样（参数“--angles 8,8,8”）。 </li><li>允许在穗视图向量的方向的16度的变化（参数“--thetaphilimit 16”），但保持周围的穗长轴线固定的（参数“--alphalimit 0”）的角度。 </li><li>让小平移的变化（ 例如 ，5像素）来解释不准确的尖峰人工采摘（参数“--shiftlimit 5”）。 </li><li>适用于高对称性（ 如 C100）的平均值（参数“--symmetry C100”），近似围绕秒杀的长轴圆柱形的平均水平。 注：对称化是用来减少在平均噪声。 </li><li>使用真正的空间屏蔽和步骤2.3和2.4生成的倒易空间面具来掩盖周围走尖峰和账户失踪的楔形（参数“--M问“和”--mask“，分别）。 </li><li>使用在2.6生成的两个MAP文件（由标记“偶”表示，并在该文件名“奇”）作为模板（参数“--Template1”和“--Template2”，分别）。 </li><li>应用低通滤波器50的分辨率（参数“--resolution”），以防止对准偏差。 </li><li>应用2的仓​​因素，加快细化（参数“--bin 2”）。 </li><li>运行5次迭代调整，平均的（参数“--firstiter 1 --lastiter 5”）。 </li></ol></li><li>第二阶段。这个阶段的目的是缩小视图四周矢量的角度。输入文件是步骤3.1.9输出SEL文件。 <ol><li>通过打开在步骤3.1.9最后一次迭代中产生的低通滤波的MAP文件测量尖峰的精确尺寸在bshow（由变量“_lp”中的文件名 ​​表示）。产生一个新的现实空间屏蔽类似于步骤2.3定义秒杀，但不包括大部分的膜和邻近的尖峰。 注：面罩的最佳尺寸取决于大小和尖峰的特征。 </li><li>用8度角取样（参数“--angles 8,8,8”）作为之前。 </li><li>允许在围绕鞋钉视图向量（参数“ - alphalimit 180”）的角180度的变化而保持的θ和披角度固定（参数“--thetaphilimit 0”）。 </li><li>不要让任何翻译班（参数“--shiftlimit 0”）。 </li><li>不适用的平均值（省略参数“--symmetry”）的任何对称性。 </li><li>使用在步骤2.4中产生的倒易空间掩模占缺失槽楔。 </li><li>使用步骤3.1.9的最后一次迭代而不对称性所产生的两个Map文件作为模板（参数R（按标签“even_nosym”，并在文件名“odd_nosym”表示）20; - template1的“和”--Template2“，分别）。 </li><li>在第一次迭代应用低通滤波器50的分辨率（参数“--resolution”），以防止对准偏差。两个独立的平均值之间自动调整，在后续的迭代滤波器参数的基础上的黄金标准傅立叶壳牌相关（FSC）（参数“--adaptivefilter”）。用0.143，标准（参数“--fsccrit 0.143”）。不允许细化过去的对比度传递函数（CTF）（参数“--minhires”）的第一个零，除非CTF校正已被施加到X线断层图。 </li><li>运行5-10重复排列和平均（ 例如 ，参数“--firstiter 6 --lastiter 15”）。监视中的变化报道的分辨率。当没有显著变化观察到的，该过程可以停止。 </li></ol></li><li>评估对称度由examini结构纳克在嵌合体 。 例如所得的低通滤波后的MAP文件（文件名 ​​中表示为变量“_lp”）时，如果道钉是三聚体复合物，3次对称性应该是显而易见的。 </li><li>第三阶段。这个阶段的目的是进一步缩小视图四周矢量的角度与正确的对称性。输入文件是步骤3.2.9输出SEL文件。的参数是相同的，在步骤3.2，只有少数例外： <ol><li>让周围的秒杀观察向量的夹角适当的修改。例如，如果该结构具有3倍的对称性，允许以60度的α角的变化（参数“--alphalimit 60”）。 </li><li>适用于平均值（参数--symmetry C3“）的正确对称（ 如 C3）。 </li><li>使用最后一次迭代生成的两个MAP文件步骤3.2.9（文件名记为标签“，甚至”与“奇”）作为输入模板文件（参数“--Template1”和 --Template2）。 </li><li>运行5-10重复排列和平均（ 例如 ，参数“--firstiter 16 --lastiter 25”）。监视中的变化报道的分辨率。当没有显著变化观察到的，该过程可以停止。 </li></ol></li><li>第四阶段。这个阶段的目的是要精确地同时缩小所有三个角度。输入文件是步骤3.4.4输出SEL文件。的参数是相同的，在步骤3.4，只有少数例外： <ol><li>允许在围绕鞋钉视图向量（参数“ - alphalimit 8”）的角8度的改变和在穗视图向量的方向8度的变化（参数“--thetaphilimit 8”）。完善的方向，以4级精度（参数“--angles 8,8,8 --iterate 2”）。 </li><li>允许较小的变化（ 比如 ，5个像素），来解释在先前的校准步骤的误差（参数“--shiftlimit 5”）。 </li><li> ü硒在步骤3.4.4最后一次迭代生成的两个MAP文件（通过标签“，甚至”表示，并在文件名“奇”）作为输入模板文件（参数“--Template1”和--Template2）。 </li><li>运行5-10重复排列和平均（ 例如 ，参数“--firstiter 26 --lastiter 35”）。监视中的变化报道的分辨率。当没有显著变化观察到的，该过程可以停止。 </li></ol></li></ol>四代种子的病毒表面的模板匹配和对齐<ol><li>位于生成均匀地涂在病毒表面的模板匹配的种子，并通过运行jviews.py分配一个初始视图向量的种子。使用步骤1.2.2输入文件生成的STAR文件。产生比尖峰的期望数目约1.5倍以上的种子。 注：这一观点矢量接近最接近每个种子点秒杀的方向。<ol><li>要产生均匀分布的种子在近似球形病毒颗粒（参数“--Even”），给半径（参数“--radius”），角距（ 如 20度）的种子（参数“--angle 20” ）和中央的病毒体的坐标。 例如 ，如果病毒体被集中（步骤1.2）中的箱具有250×250×250个像素的大小之后，中央坐标是125125125像素（选项“--origin 125125125”） 。 </li><li>要生成的丝状颗粒均匀分布的种子，使用参数“--Filament”，并同时指定半径和螺旋对称参数（上升和扭曲）。 注：该螺旋对称的参数在这里只是作为界定均匀分布的种子位置的一种便捷方式，他们并不需要反思灯丝尖峰的实际顺序。 </li></ol></li><li>产生的病毒表面explaine两个独立的平均值d在第2节。 <ol><li>产生SEL文件中定义属于集合“1”和“2”使用jsubtomo_evenodd.py和步骤4.1生成的星病毒体文件。 注：为确保数据集的独立性，以前设置的病毒颗粒成团“1”和“2”必须保持不变。 </li></ol></li><li>完善种子的位置。 <ol><li>按照3.1的步骤说明，除非另有说明。使用步骤4.2.1作为输入文件生成的SEL文件。 </li><li>让种子沿法线仅转移至膜（参数“--zshiftlimit”）。调节移位的容许量取决于病毒颗粒从理想球面几何多少偏离（ 例如 ，参数--zshiftlimit 25“）。 </li><li>使用步骤4.2生成的两个MAP文件作为输入模板文件（参数“--Tem（由标签”偶“，并在文件名”奇“表示）板1“和--Template2）。 </li><li>采用独特的后缀，以避免覆盖原输入之星文件（ 例如 ，参数“--suffix _seeds”）。 </li><li>使用的4像素合并以加速计算（参数“--bin 4”）和一个低通滤波器（ 例如 ，参数“--resolution 50”）。 </li></ol></li><li>使用jviews.py（参数“--cmm”）产生精种子之星档案奇美拉标记文件（CMM）。检查种子在嵌合体打开了CMM文件（以及相关的病毒MAP文件）。确保成品种子正确相对于病毒膜对齐。 注意：不同的颜色可以被用于区分标记物的单独套（参数“--color”）。备选地精制标记可以基于它们的交叉相关系数（ 例如 ，参数“--fomcolor 0.1,0.3”）进行着色。 </li></ol> 5，黄金展位ARD迭代对齐和穗粒结构的平均自动找到附近使用精致的瓜子本地模板匹配的病毒分卷的所有尖峰和对齐和平均位于尖峰。使用从手工采摘尖峰初始模板的一个子集所产生的平均水平。 <ol><li>围绕种子进行局部模板匹配以平均使用的所有程序jsubtomo_iterate_gold.py的尖峰。按照步骤3.5的说明，除非另有说明。 <ol><li>输入文件是在步骤4.3生成的种子提炼的SEL文件。 </li><li>为视图矢量角用一个足够大的限制。 例如 ，如果种子产生每20度（步骤4.1），使用的角度大于该值的一半稍大（参数“--thetaphilimit 12”）。让周围的秒杀观察向量的夹角适当的修改。 </li><li>让种子在所述膜的平面内移动（参数“--xyshiftlimit”）。 例如 ，如果种子到种子的距离是25个像素，使用总变速比一半的（ 例如略多，参数“--shiftlimit 6 --xyshiflimit 10”）。 可能需要额外的翻译转移，如果在步骤3.5生成的地图文件都集中在离中心病毒比种子不同的距离：注意。 </li><li>使用步骤3.5生成的两个MAP文件（通过标签“，甚至”表示，并在文件名“奇”）作为输入模板文件（参数“--Template1”和--Template2）。 </li><li>使用步骤3.5（参数--resolution）生成映射文件的当前的分辨率。 </li><li>运行5-10重复模板匹配，对齐和平均（ 例如 ，参数“--firstiter 1 --lastiter 10”）。监视中的变化报道的分辨率。当没有显著变化观察到的，该过程可以停止。 </li><li>每次迭代后，排除重叠的点击率。 例如 ，如果尖峰的宽度为30像素，排除命中是接近了30个像素，以其他的打击（参数“--mindist 30”）。 </li><li>排除安打与低互相关合作效率。例如，包括最好的75％的尖峰对于每个病毒粒子（参数“--topp 75”）。 注：点击数与低互相关合作效率很可能代表假阳性命中。 </li></ol></li></ol> 6，可视化的结果<ol><li>在嵌合体可视化开启秒杀的精致结构和装修的原子结构。 <ol><li>使用在最后一次迭代产生的低通滤波后的MAP文件（用标记“LP”），在步骤5.1.6作为输入文件。 </li><li>使用基于在嵌合体装修交相关步骤5.1.6所示的决议“适合的地图”的工具。 </li></ol></li><li>创建virio的复合模式N使用jsubtomo_create_model.py。 <ol><li>使用在最后一次迭代产生的低通滤波后的MAP文件（用标记“LP”），在步骤5.1.6作为输入的MAP文件（参数“--template”）。 </li><li>在步骤5.1.6作为输入之星文件使用在最后一次迭代产生之星文件。 </li><li>使用步骤3.2.1生成的模板文件占的复合模式（参数“--mask”）重叠的密度。 </li><li>表明病毒粒子MAP文件的大小，以产生相同的大小的复合模型（参数“--size”）。 </li></ol></li><li>打开复合模型进行可视化的嵌合体 。 </li></ol>

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The authors have nothing to disclose.

<html>知识的病毒糖蛋白的尖峰结构对病毒体膜是用于理解病毒的复制和显影疗法，治疗和预防感染是必不可少的。电子低温显微术结合单个颗粒的平均已成为使用最多的方法来解决包膜病毒粒子，包括糖蛋白的尖峰的结构。然而，该方法仅限于icosahedrally对称的病毒。在这里，通过低温电子断层扫描和subtomogram平均在Jsubtomo的应用程序，我们已经提出了一个通用协议，用于确定糖蛋白尖峰形性包膜病毒是不适合其他流动结构生物学的方法。我们的代表性结果证明，该方法的分辨率足以揭示见解域架构，低聚，并在完整的病毒颗粒的糖蛋白尖峰高阶组织。 jove_content“&gt;这个协议中最关键的一步是兴建两座可靠的启动模式，在统计上是相互独立的。成功执行此步骤假定糖尖峰足够大，不装反目成仇得太紧，使个人尖峰在视觉上识别和手动拾取的断层图像，以及两个独立的模式取平均。如果这是不可行的，两个修饰的协议可以尝试。首先，两个独立的随机模型可通过首先定义subtomograms两个随机子集，然后构造这些子集30内平均该subtomograms。其次，如果孤立穗的结构是来自通过其他手段，例如通过X射线晶体结构，它可以被用作初始模型，然而，必须小心到低使用低分辨率截止（50-70）带通滤波器这种模型中，如产生的两个模型在下一回合的细化将是统计独立只有超过这个分辨率。由于这个警告，前一种方法的建议。 从该协议中获得的分辨率取决于四个主要因素：1。数据收集策略和所述输入数据的质量，ⅱ。该subtomograms的数目，三。该subtomograms的对准精度，以及iv。异质性的结构。而第一和第二限定可以在很大程度上克服通过使用高的信号 - 噪声直接电子检测器结合的CTF校正成像和自动数据采集，对准精度进一步受到关注自身结构的大小和形状。当应用该协议上缺乏显着的特征的小尖峰，它可能是有利的Fab片段结合于尖峰提高对准精度，因而分辨率31。最后，如​​果结构被平均展出多个构象，子tomogr时的分类方法可用于分别平均不同的构象。为此，Jsubtomo集成了发电机包，提供强大subtomogram分类9。 上述协议是互补的分离的病毒糖蛋白的X射线晶体结构。晶体结构可以被嵌入到子断层图像的平均值，得到的糖蛋白的精确取向相对于所述病毒体膜。这种方法的应用，无疑将继续脱落到包膜病毒的结构和病理学的光。

电子冷冻断层扫描是一种电子低温显微成像技术允许一个三维（3D）重建的复杂生物样品的计算。合适的样本范围从纯化的大分子复合物1中 ，单丝2，被小泡3，和多形性膜病毒4至整个原核细胞5，甚至薄整个真核细胞6的区域。以下的倾斜系列，3D断层扫描卷或断层图像的数据收集，可以使用几种建立软件包，包括Bsoft 7和IMOD 8来计算。 固有的生物标本通过电子低温断层限制相应的断层卷的生物学解释的研究两个方面。首先，由于可应用到生物材料引入显著辐射损伤之前的有限的电子剂量，Signa的升噪比在断层数据通常是非常低的。其次，由于数据收集过程中有限样本的倾斜几何的结果，一些意见的对象仍然存在，导致在断层容积所谓的“缺少楔”神器。然而，无论这些限制可以如果断层容积包含重复相同的结构，如大分子复合物，可以成功地平均9-12来克服。 之前，均从断层图像的重建结构中，感兴趣的对象，必须找到并对齐，以相同的方向。定位这种结构可以通过一个模板结构中的断层容积的互相关使用通常被称为模板匹配13的方法来实现。在这个匹配过程中使用的模板可以从电子低温显微术或电子低温层析结合3D重建中得到，或者它可以是从模拟的密度图原子结构。一些计算软件包已经开发完成这些任务11。 平均膜病毒，如HIV-1糖蛋白尖峰，一直是特别成功的方法来研究它们的结构14-16。该结构的理解是积分揭示病毒 - 宿主相互作用的两个分子基础和指导抗病毒和疫苗的设计开发。而大分子晶体是首选的高分辨率个别病毒糖蛋白及其复合物的结构分析（通常大于4埃更好）的技术中，从该方法中产生的X射线结构是蛋白质的天然膜环境中分离的病毒颗粒的。因此，重要的信息，如病毒糖蛋白的高次结构，在病毒粒子的情况下，仍然缺乏。另一方面，电子低温显微术和单粒子重建的整个包膜病毒被限制到病毒粒子具有二十面体对称17,18。电子cryotomography结合分卷排列也因此成为一个互补的技术，允许形状不规则， 在原地多形病毒糖蛋白尖刺的研究。 我们已经开发了一个名为Jsubtomo（www.opic.ox.ac.uk/jsubtomo）的断层分卷的检测，校准和平均软件。 Jsubtomo已被用于一些细胞和病毒结构19-26的结构测定。在这里，我们勾勒出一个详细的协议，这使得测定病毒表面刺突结构。要通过相关的噪声规避过度细化的平均结构中，“黄金标准”细化方案采用10,27。最后，对于可视化的典型结果的解释策略进行了讨论。

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jsubtomo (ver 1.3.1)</td>
			<td style="width:184px;">University of Oxford</td>
			<td style="width:119px;">n/a</td>
			<td>www.opic.ac.uk/jsubtomo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bsoft (ver 1.8.7)</td>
			<td style="width:184px;">NIAMS, NIH</td>
			<td style="width:119px;">n/a</td>
			<td>bsoft.ws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UCSF Chimera</td>
			<td style="width:184px;">UCSF</td>
			<td style="width:119px;">n/a</td>
			<td>www.cgl.ucsf.edu/chimera</td>
		</tr>
	</tbody>
</table>

averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using jsubtomo

包膜病毒利用在其表面上的膜糖蛋白介导进入宿主细胞。这些糖蛋白&#39;尖峰&#39;的三维结构分析通常是在技术上具有挑战性，但重要的是了解病毒的发病机制和药物设计。在这里，一个协议提交病毒刺突结构的决心，通过低温电子断层扫描数据计算平均值。低温电子断层扫描是电子显微镜技术来获得三维断层容积重建，或断层，多形性生物样本，如在接近本机，冷冻水合状态的膜病毒。这些断层图像揭示的利益结构在三维空间中，虽然在低的分辨率。子体积，或子断层图像的计算平均值，是必要的，以获得重复的结构基序，如病毒糖蛋白的尖峰的更高分辨率的细节。阿里的详细计算方法gning和平均使用Jsubtomo软件包子断层概述。这种方法使病毒糖蛋白的尖峰结构的可视化高阶尖峰对尖峰上的病毒膜相互作用的研究在20-40范围内的分辨率和研究。典型的结果示于Bunyamwera病毒，从家庭布尼亚病毒科的包膜病毒。该系列是病原体威胁到人类和动物的健康构成威胁一个结构多元化的群体。

我们证明上面使用此前公布的数据Bunyamwera病毒（Orthobunyavirus， 布尼亚病毒科 ）的包膜糖蛋白复合物中列出的子断层扫描平均工作流程设置24应用程序。数据收集和精化参数列于表1中 ，其中一个代表断层图像示于图2。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 参数（单位） </td><td> 价值 </td></tr><tr><td> 数据收集 </td><td></td></tr><tr><td>电压（kV） </td><td> 300 </td></tr><tr><td>校准放大倍数（倍） </td><td> 111,000 </td></tr><tr><td>像素尺寸（） </td><td> 5.4 </td></tr><tr><td>估计量（E - / 2） </td><td> 100 </td></tr><tr><td> Underfocus（微米） </td><td> 4.0-4.5 </tD&gt; </tr><tr><td>首先周大福零（一） </td><td> 26-30 </td></tr><tr><td>倾斜范围（°） </td><td> -60-60 </td></tr><tr><td>倾斜采样（°） </td><td> 3 </td></tr><tr><td> 数据和细化 </td><td></td></tr><tr><td>断层</td><td> 11 </td></tr><tr><td>病毒体</td><td> 29 </td></tr><tr><td>每个病毒种子</td><td> 106 </td></tr><tr><td>种子总数</td><td> 3074 </td></tr><tr><td>尖峰检测</td><td> 1,346 </td></tr><tr><td>除去重叠后，尖峰</td><td> 1,401 </td></tr><tr><td>互相关为基础的选择后的尖峰</td><td> 1,022 </td></tr><tr><td>包括在最终的平均尖峰</td><td> 1,022 </td></tr><tr><td>对称</td><td> C3 </td></tr><tr><td>角度采样（°） </td><td> 8 </td></tr><tr><td>分辨率在细化使用范围（A） </td><td> 42-334 </td></tr><tr><td>最后的解决估计（A）B </td><td> 35 </td></tr></tbody></table> 表1：Bunyamwera数据收集和细化的统计数据。 à周大福，对比传递函数。 b。使用在0.143阈值两个独立的精致结构之间的傅立叶壳的相关计算。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> 图2：通过Bunyamwera病毒体的断层图像切片中的每个病毒粒子的外周明显若干尖峰的侧视图来表示带箭头。的断层图像已经经过低通滤波，以60埃。比例尺为100nm。 首先，我们使用精制205人工采摘尖峰初始模型（ <strong&gt;图3）。最中心的尖峰的三次对称性是不施加任何对称性（ 图3B）明显和被强加在随后的几轮细化（ 图3C）。对于病毒粒子表面上自动地检测所有的尖峰，我们产生106种对每个病毒粒子在43 nm和20度（ 图4A）的间距半径和迭代细化它们的位置相对于所述膜（ 图4B）。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> 图3：起始模板结构的细化。 （一）圆柱的平均尖峰手动定义的位置建造（C100）模板（B）平均为密度经过五轮的细化，没有任何对称（C）施加显示器秒杀了三倍对称的特点。该模型的分辨率为48。穗（三）平均在41后的五轮细化了三次对称性解决。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> 图4：将种子细化AB）的种子之前（A的一个子集）和之后（B）的细化是从图2所示的一个病毒粒子密度在（B）的种子已经颜色编码基于各个交叉。相关系数（蓝色，低相关性;红色，高的相关性）。 最优相关糖蛋白刺突补丁（顶部75％的去除重叠后;〜1,000尖峰）被用于计算最终的平均值。平均得到解决，以35的分辨率（ 图5）。它揭示了三聚尖峰结构的中间，在除了从六个邻近的尖峰一定的贡献。的病毒粒子，通过将结构中的已知位置来计算的复合模型，显示在病毒粒子表面上（ 图6A）中的尖峰的位置。偶尔，尖峰局部有序的补丁是很明显的（ 图6B）。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> 图5：模板匹配后的糖蛋白刺突层的贴片精致结构。 （AB）两地图“偶”和“奇”，从数据的两半重建示。这两个图显示一个显着的相似程度，验证了该方法的合法性验证。围绕穗长轴线的方向是不同的两个映射已经重建完全彼此独立。这两个地图（C）平均显示在35埃的最终解决。 <img alt="图6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> 图6：在Bunyamwera病毒尖刺的放置。 （一）病毒体的复合模式。查看向量（支）表示尖峰的方向。颜色表示每个穗和模板结构（蓝色，低相关性;红色，高相关）之间的互相关（B）一个特写尖峰的有序贴剂。

Watch this Scientific Journal Video about Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo at JoVE.com

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

averaging-viral-envelope-glycoprotein-spikes-from-electron

Research

JoVE Journal

Immunology and Infection

12.0K 次观看.  University of Oxford. An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

视频: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

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 ...

科研

免疫与感染

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

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 ...