该项目的经费部分来自国家反应和传染性疾病研究所、国家卫生研究院、卫生和人类服务部的联邦基金 , CEIRS 合同 HHSN272201400008C ( 胡法光 ) ;NIH U19AI109946-01 (胡法光);和 P01AI097092-04S1 (P.E.L.)。

警告: 在人类人口中传播的一些流感病毒 (如 、H1、H3) 是生物安全等级2级的病原体, 必须用护理和适当的个人防护设备来处理。病毒的处理必须得到机构审查委员会的批准。下列议定书已由西奈山的机构审查委员会批准. 
 注意: HA 特定的抗病毒复制的抗体通常可以归类为 i), 它们绑定在球状头和 ii 的受体结合位点上或相邻的部位, 并结合受体束缚的远端。域, 包括球形头的侧面和 HA 的茎区。靶向受体结合位点的抗体可以防止唾液酸图案在靶细胞表面的接触, 并可通过血凝抑制 (HI) 测定来测定。抗体是高阴性的, 如茎特异抗体, 仍然可以抑制病毒复制, 但只能使用中和化验来评估. 
 1. 基于 hi 和中和活动的抗体分类 <ol> <li> hi 化验 <ol> 在96井 V 底板中, 添加25和 #181; 1x PBS 在2到12列中的 L. <li>
		<li> 稀释 7B2 (特定于头), 6F12 (特定柄) 23 和一个同种控制到100和 #181; 在 1x PBS 和分50和 #181;还包括一个无单克隆控制通过添加50和 #181; L 1x PBS ( 图 1A ). </li>
		<li> 通过传输25和 #181, 从1列到2列中执行2倍的抗体序列稀释, 依此类推。丢弃最后25和 #181; L 从12列 ( 图 1A ). 
		注意: 请确保包含一排无抗体控制的行. </li>
		<li> 稀释病毒存量 (一种重组病毒, 表达 A/California/04/09 与 Rico/8/34 的内部片段的 HA 和 NA) 到8血凝 units/25 和 #181; 我稀释了病毒的库存添加25和 #181; 稀释后的病毒储存 (8 血凝) 到每一个井 (A 排到 G). 
		注: 抗体和病毒混合物应具有50和 #181 的最终体积; L 具有起始最终浓度50和 #181; g/毫升. </li>
		<li> 室温 (RT) 孵育板45分钟 </li>
		<li> 对于后滴定排 (H), 增加50和 #181; L 1x PBS 在井 H2 到 H12。增加100和 #181; 8 血凝 units/25 和 #181 的 l; H1。通过转移50和 #181 连续稀释二倍; L 从 H1 到 H2, 等等。丢弃最后50和 #181; 我来自 H12最后, 添加50和 #181; L 0.5% 鸡红细胞 (RBC) 的所有水井的96井 V 底板. 
		注: 检测中的单克隆抗体样本应具有100和 #181 的最终体积; l: 单克隆 (25-#181; l), 病毒 (25 和 #181; l) 和红细胞 (50 和 #181; l)。无单克隆人控制的最终体积应包含25和 #181; 1x PBS、25、#181、病毒 l 和50和 #181; 红细胞 l. </li>
		<li> 在4和 #176 上孵育盘; C 为 1 h. </li>
		<li> 视觉阅读板的高活动。如果有一个特定抗体的阳性读数, 请继续步骤2.1 以产生逃逸变种。如果抗体是高阴性, 继续下面到步骤 1.2, 以评估是否抗体有中和活性的细胞培养法. 
		注: 高活性单克隆的阳性读数由一个井中心的暗红色红细胞颗粒 ( 图 1B ; 7B2 ) 表示, 当96井 V 底板在45和 #176 上举行时, 将形成撕裂下降; 角度。负读数不会在井中形成暗红色的红细胞颗粒 ( 图 1B ; 6F12 , 没有单克隆)。同种控制也不会形成一个暗红色的红细胞颗粒, 应该看起来相同的茎特异抗体, 6F12 或无单克隆控制样本 ( 图 1B ) 23 . </li>
	</ol> </li> <li> Microneutralization 检测 <ol> <li> 板 Madin 在一个密度为 2 x 10 4 细胞/良好的组织培养治疗 96-井板和孵育37和 #176; C 和 5% CO 2 17 到 19 h. 
		注: 在使用前, 电池还可以保持在井下至少4小时. </li>
		<li> 在一个单独的96井板中, 执行七3倍的人类单克隆稀释 4D05 5 , CR9114 17 或同种 IgG 控制, 在起始浓度为200和 #181; 1X 最小基本介质 (记忆) 中的 g/毫升, 辅以磺phenylalanyl 氯甲基酮 (TPCK)-处理的胰蛋白酶 (1 和 #181; g/毫升) ( 图 2 ). 
		注: 排 A 应包含75和 #181; 稀释抗体 (起始浓度为200和 #181; g/毫升)。连续稀释 (3 倍) 下板通过转移25和 #181; L 从 a 排到 B 排, 等等。a 到 H 的行的最终音量应为50和 #181;不需要在稀释转移之间改变技巧. </li>
		<li> 稀释病毒储存 (重组病毒表达 HA 和 NA 的 A/Shanghai/1/13 与内段的 a/波多黎各 Rico/8/34) 到100的50% 组织培养感染剂量 (TCID 50 )/50 和 #181; L 在1x 内存补充与 TPCK 治疗胰蛋白酶 (1 和 #181; g/毫升) 24 。添加50和 #181; 将稀释病毒的 l-/井的抗体准备 (步骤 1.2.2)。添加50和 #181; L 1X 记忆到未受感染的细胞控制井. </li>
		<li> 孵育病毒抗体混合物在37和 #176; C 孵化器 (与 5% CO 2 ) 为 1 h. 
		注: 病毒抗体混合物的总体积应为100和 #181; l:50 和 #181; 抗体稀释 (步 1.1.2) 和50和 #181; l 病毒包含 100 TCID 50 (步骤 1.2.3). </li>
		<li> 在井中吸进介质, 并将整个100和 #181 的病毒抗体混合物添加到相应的井中. 
		注意: 这种吸入是用一个连接到真空的8通道器适配器完成的。另外, 8-或12多通道微移液管可用于人工抽吸。接种去除或洗涤过程中的所有吸入都是从抗体的最高浓度降到最低, 而无需改变提示. </li>
		<li> 200 和 #181 冲洗单层; 1x PBS 的 L。吸气200和 #181; L 1x PBS (如步骤 1.2.5)。重复洗涤一次, 共洗两次. </li>
		<li> 通过添加整个100和 #181 感染 MDCK 细胞单层; 病毒抗体混合物 (从台阶 1.2.4) 在单层上, 感染/孵育在37和 #176; C (5% CO 2 ) 为 1 h. </li>
		<li> 在感染期间, 在一个单独的96井板中, 准备另一套抗体稀释。添加150和 #181; 100 和 #181 的 L; 每排 a 和100和 #181 的各自抗体的 g/毫升; 1x 记忆补充 TPCK 处理的胰蛋白酶 (1 和 #181; g/毫升) 在 b 行通过转移50和 #181 执行3倍稀释; l 从 a 行到 b 行, 等等, 下到行 h 放弃最后50和 #181; L 从列 h。每一个井的总体积应等于100和 #181; l. 预留. 
		注: 抗体在1x 的记忆中被稀释, 辅以 TPCK 处理的胰蛋白酶 (1 和 #181; g/毫升). </li>
		<li> 从单层1.2.7 中吸取病毒抗体接种, 并与整个100和 #181 进行补充; 在步骤1.2.8 中制备的适当抗体稀释液的 l-/井. 
		注: 如果一个井包含最终抗体浓度100和 #181; 在感染 (步骤 1.2.7), 然后补充培养基也应包含最终抗体浓度100和 #181; g/毫升 (步骤 1.2.8). </li>
		<li> 在37和 #176 中孵育24小时; C 孵化器 (5% CO 2 ). </li>
		<li> 吸出96个井板上的介质, 用200和 #181 冲洗; 1x PBS 三次. </li>
		<li> 用100和 #181 固定单元格, 80% 丙酮为1小时,-20 和 #176; C. 
		注: 80% 丙酮溶液稀释在双蒸馏 (dd) H 2 O ( 例如 , 80 毫升100% 丙酮加上20毫升的 ddH 2 0)。在使用前, 80% 丙酮溶液可以在冰上冷却. </li>
		<li> 使用200和 #181 清洗单元格; 1X PBS 三次. </li>
		<li> 用200和 #181 阻挡板; 在 1X PBS 中稀释5% 牛奶, 在 RT 中孵育1小时. </li>
		<li> 添加100和 #181; 化流感核 (NP) 主抗体稀释 1:2, 000 在 1x PBS/1%bovine 血清白蛋白 (BSA) 和孵育板在 RT 为 1 h. 
		注意: 对于 b 型流感病毒, 应使用一种流感病毒特异的抗 NP 抗体. </li>
		<li> 三次用 1x PBS 冲洗盘子. </li>
		<li> 添加100和 #181; 亲和-马萝卜过氧化物酶 (HRP) 共轭抗体稀释 1:3, 000 在 1x PBS/1%bsa 和孵化的板在 RT 为1小时. </li>
		<li> 200 和 #181 清洗板; 1x PBS 三次. </li>
		<li> 在100和 #181 添加 HRP 衬底试剂; L/良好, 并在黑暗中孵育 RT. 
		注意: 在添加酸性停止缓冲液之前, 应先优化孵化时间。一般来说, 15 到30分钟就足够了. </li>
		<li> 用50和 #181 淬火反应; 5 米的盐酸. 
		注意: 5M 盐酸是一种腐蚀性很强的试剂, 可以对眼睛、皮肤和粘膜造成损害。添加此试剂应在通风罩下进行, 并配备适当的个人防护设备. </li>
		<li> 阅读 492 nm 的板块, 减去背景 (未受感染的细胞) 井. </li>
		<li> 用以下公式计算百分比抑制: 
		<img alt="Equation" src="/files/ftp_upload/56067/56067eq1.jpg"/> </li> <li> 如果抗体具有中和活动 (且没有 HI 活动), 请继续执行步骤 2.2. 
		注意: 缺乏 体外 中和活动的抗体将缺乏 HI 活性. </li>
	</ol> </li> </ol> 2。转义变种变体的生成 注意: 对具有或缺少 HI 活动的中和抗体进行进一步分析, 并使用下面描述的特定协议. 
<ol> <li> 协议 1: HI 阳性/中和阳性抗体 ( 图 3A ) <ol> <li> 在 1x PBS 中准备一组 10 6 斑块成型单位/毫升 (PFU/毫升) 的病毒储存400和 #181; L 音量. </li>
		<li> 在 1x PBS 中, 在0.005 和 #181 的容量中准备四稀释的感兴趣的抗体浓度 (如 、0、0.5、0.05 和100毫克/毫升); 每次稀释. 
		注意: 野生型病毒应始终传代, 并在缺乏抗体。这些传代病毒的序列将有助于区分适应细胞培养条件和逃逸突变. </li>
		<li> 混合100和 #181; l 10 6 PFU/毫升病毒与100和 #181; 每个抗体稀释或100和 #181; l 1x PBS. </li>
		<li> 在37和 #176 中孵育1小时; C 孵化器 (5% CO 2 )。漩涡简要地。将每种混合物的200和 #181 注入特定的无病原体 (SPF) 胚鸡卵. </li>
		<li> 将卵孵化为37和 #176; C (不含 CO 2 ) 用于 40-44 h. </li>
		<li> 通过在4和 #176 中孵化来牺牲感染病毒的胚卵; C 至少为 6 h. </li>
		<li> 从鸡蛋中获取囊流体, 如前所述 24 , 25 . </li>
		<li> 执行血凝试验, 如上所述 24 , 26 。如果所有的囊液体制剂没有血凝滴, 重复从步骤2与抗体稀释范围从0.005 毫克/毫升到0.00005 毫克/毫升. 
		注: 高阳性抗体的饱和浓度可以抵消所有存在的病毒微粒。因此, 可能有必要减少传代中存在的抗体量. </li>
		<li> 通过执行 HI 检测 24 (步骤 1.1) 来确认转义变体. 
		注: 高活性抗体阻滞 HA 在靶细胞上参与唾液酸的主题。因此, 一个病毒在其同源抗体的存在失去了能力, 凝集红细胞 (存在红细胞颗粒)。理论上, 高活性抗体的逃逸变种仍然可以结合唾液酸的主题, 即使在其同源抗体的存在, 从而可以凝集红细胞 (无红细胞颗粒)。如果感兴趣的抗体的 HI 仍然是可检测的, 重复的协议从步2.1.2 以更高的起始集中抗体. </li>
	</ol> </li> <li> 协议 2: hi-阴性/中和-阳性抗体 ( 图 3B ) 注: 为了产生逃逸变种对抗中和抗体缺乏 HI活动中, 病毒必须在传代的情况下出现, 增加抗体量。
	<ol> <li> 在 1 x 10 6 单元格的密度下 MDCK 单元格, 并在4和 #176 中孵育至少 37 h; C 孵化器 (与 5% CO 2 )。
		<li> 将病毒的库存稀释到 10 6 PFU/毫升或前一段中的病毒, 在1x 内存中使用 TPCK 处理的胰蛋白酶 (1 和 #181; g/毫升) 在500和 #181; L 音量. </li>
		<li> 在250和 #181 中, 准备一个单一稀释的抗体 (0.02 毫克/毫升, 用于原通路或更高的所有后续通道), 在 TPCK 处理后的胰蛋白酶中进行 1x; L 体积. </li>
		<li> 混合250和 #181; 稀释后的病毒的 l, 250 和 #181; 稀释抗体 (+ 抗体) 或250和 #181; l 1x 记忆 (无抗体控制). </li>
		<li> 在37和 #176 中孵育病毒抗体混合物30分钟; C 孵化器 (5% CO 2 ). </li>
		<li> 使用玻璃巴斯德吸管吸出介质, 用1毫升的 1X PBS 清洗细胞单层. </li>
		<li> 添加500和 #181; L 将混合物放入井中, 在37和 #176 中孵育; C 孵化器 (5% CO 2 ) 为 1 h. </li>
		<li> 1 小时后, 用 TPCK 处理的胰蛋白酶 (1 和 #181; g/毫升), 补充2毫升1x 记忆的水井. </li>
		<li> 在显微镜下检查 48 h 感染的细胞是否有病变效应 (CPE) 的迹象, 或进行血凝检测以检测病毒生长情况 26 . </li>
		<li> 如果有总 CPE 在文化补充与抗体, 收获上清在多个低温管, 标签与段落数字和商店在-80 和 #176; C. </li>
		<li> 保存100和 #181; 升上清液, 用2毫升的1x 记忆 MDCKs TPCK-胰蛋白酶和抗体来感染新鲜的单层膜。记住, 包括一个没有抗体控制每一个段落. 
		注: 在下一篇文章 (每两天) 中增加两倍 (或由研究员斟酌决定) 的抗体浓度。</li> <li> 增加每个连续通道中抗体的浓度, 直到病毒生长仍然可行, 即使最终浓度为0.6 毫克/毫升抗体。冻结每条通道的上清液瓶, 并贮存于-80 及 #176; C. 
		注意: 无抗体控制对于验证病毒从一个通道到另一段的增长是至关重要的。如果有总 cpe 在无抗体控制, 但没有 cpe 在 + 抗体组, 这表明, 抗体浓度太高, 并没有逃脱变种产生。如果有总 cpe 在没有抗体控制, 但仅适度 cpe 在 + 抗体小组, 这表明存在潜在的逃命变异。在下一段中, 增加通道体积到200和 #181; L 上清和保持抗体的浓度, 以增加产生逃逸变种的可能性. </li>
	</ol> </li> </ol> 3。通过斑块纯化分离逸出变形 <ol> <li> 在 1 x 10 6 单元格中的密度为6个的板中 MDCK 单元格, 并在4和 #176 中孵育至少 37 h; C 孵化器 (与 5% CO 2 )。
	<li> 将 1x TPCK 中的抗体稀释到300和 #181; 在250和 #181 的体积内进行 g/毫升; l 与250和 #181 混合; 与相应的逃逸突变病毒的 l。病毒传代在缺乏抗体的同时也应进行菌斑纯化. </li>
	<li> 从细胞中吸取介质, 用 1x PBS 三次冲洗, 并添加整个500和 #181; 病毒抗体混合物的 L (步骤 3.2). </li>
	<li> 在37和 #176 中孵育1小时的板; C 孵化器 (与 5% CO 2 ) 确保每10分钟来回晃动以防止单层干燥. </li>
	<li> 吸出病毒抗体混合物, 并用含有相应数量抗体的覆盖琼脂培养基 (300 和 #181; g/毫升; 步骤 3.2) 补充水井. </li>
	<li> 在37和 #176 中孵育40-44 小时的板; C 孵化器 (5% CO 2 ). </li>
	<li> 将可见的斑块与蓝色或黑色的标记圈在一起以方便斑块采摘. </li>
	<li> 在没有抗体的情况下, 为每个逃逸突变病毒抗体组合选择四斑块, 以及在 MDCK 细胞或卵中传代的野生型病毒. </li>
	<li> 重100和 #181 的牌匾; 1x PBS 的 L. </li>
	<li> 注入整个100和 #181; 将斑块纯化后的病毒转化为10天的 SPF 胚鸡卵. </li>
	<li> 在37和 #176 中孵育40-44 小时的卵; C 孵化器 (不含 5% CO 2 ). </li>
	<li> 执行血凝试验以确认病毒的存在 (步骤 1.1). </li>
</ol> 4。病毒 rna 的提取及 HA 序列变异的分析 <ol> <li> 从200和 #181 中提取病毒 rna; 逸出突变体病毒囊液与苯酚和异硫氰酸胍的单阶段性溶液. 
	注意: 苯酚是一种挥发性液体试剂, 可引起咳嗽、气短, 并通过接触适度刺激皮肤. </li>
	<li> 将 ha 段从病毒 RNA 中放大, 并使用反转录酶和基因特异的引物为 ha 段 27 . 
	注: 表2中描述的乙型流感病毒的通用引物放大了 HA (~ 1800 bp) 和 NA (~ 1500 bp) 28 . </li>
	<li> 在1% 琼脂糖凝胶中解决 RT PCR 产品, 并切割出正确尺寸的波段 (~ 1800 bp). </li>
	<li> 凝胶提取 PCR 产品采用硅胶膜为基础的纯化程序, 并发送 cDNA 的排序. </li>
	<li> 识别抗体结合所需的氨基酸残留物, 区分假定的逃逸变种和传代野生型病毒的突变, 这是由于细胞培养适应或免疫选择. </li>
	<li> 将包含野生类型或转义变体 HA 的 PCR 产品克隆为 pCAGGs 表达载体 (NotI 和 NheI). </li>
	<li> 对转义变体 HA 的抗体绑定可以用下面描述的两个选项之一来评估. </li>
</ol> 5。转义变体的抗体结合分析 <ol> <li> 免疫荧光 <ol> <li> 板293T 密度为 2 x 10 4 细胞/井在 96-井板和孵化在37和 #176; C 孵化器 (与 5% CO 2 ) 为 24 h。</li> <li> 染0.10 和 #181 的单元格; pCAGGS 质粒编码逃逸突变 ha, 病毒传代 ha, 和野生型 ha (unpassaged) 与使用转染试剂. </li>
		<li> 在37和 #176 中孵育 48 h 的96井板; C 孵化器 (5% CO 2 ). </li>
		<li> 用100和 #181 进行修复; 0.5% 甲醛为15分钟的 RT. 
		注意: 甲醛是一种挥发性液体试剂, 可引起咳嗽、气短, 并通过接触适度刺激皮肤。它被指定为一种潜在的人类致癌物。添加试剂应在通风的化学罩中进行. </li>
		<li> 使用 PBS 1x 冲洗三次。在 RT 的 1x PBS 中用5% 的牛奶块1小时. </li>
		<li> 使用 1x PBS 清洗三次。孵育与5和 #181; 1 小时的抗体的 g/毫升在 RT. </li>
		<li> 孵育100和 #181; 第二抗体 (人或 anti-mouse Alexa 488) 在稀释 1:2, 000 在 1x PBS/1%bsa 为 1 h 在 RT 在黑暗中. </li>
		<li> 使用 1x PBS 清洗三次。在荧光显微镜上观察细胞阳性或阴性的染色. </li>
	</ol> </li> <li> 荧光激活的单元格排序 (资产处理) <ol> <li> 板293T 单元格的密度为 2 x 10 5 单元格/井在6孔板上, 孵育在37和 #176; C (与 5% CO 2 ) 为 24 h. </li>
		<li> 染0.50 和 #181 的单元格; g/与 pCAGGS 质粒编码逃逸突变 ha, 病毒只有传代 ha, 和野生型 ha 与使用转染试剂. </li>
		<li> 在37和 #176 中孵育 48 h 的6井板; C (5% CO 2 ). </li>
		<li> 48 小时后, 吸气培养基, 用 1x PBS 轻轻冲洗两次 (确保单层不受干扰). </li>
		<li> 获取转染的293T 细胞与500和 #181; L 的外地资产管制署缓冲区 (1x PBS/2%fetal 小牛血清). 
		注: 在使用前, 应 pre-chilled 4 和 #186; C. </li>
		<li> 将收获的293T 细胞在 300 x g 离心5分钟在4和 #176; C. </li>
		<li> 吸重的缓冲区, 并与200和 #181; L 的资产管制的缓冲区包含抗体的利益 (最终集中1至5和 #181; 克/毫升)。在 RT 中孵育20分钟 </li>
		<li> 在4和 #176 离心 300 x g 的细胞5分钟; c. 用500和 #181 清洗两次;用玻璃巴斯德吸管仔细吸气, 以不扰乱颗粒. </li>
		<li> 重与200和 #181; 包含二级抗体的国际资产管制的缓冲区, 含有与 Alexa 488 (最后稀释 1:1, 000)。在黑暗中孵育4和 #176; C 为 20 min. </li>
		<li> 在4和 #176 离心300克的细胞5分钟; c. 用500和 #181 清洗两次; 在外地资产管制处, 并仔细吸取洗涤缓冲器. </li>
		<li> 重500和 #181; 抗体和/或多克隆的绑定血清对转染 HA 的细胞. 
		注意: 记住在实验中包括一个无单克隆/多克隆血清控制以及一个 untransfected 样本. </li>
	</ol>
	</li>
</ol>

<ol>
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作者声明没有利益冲突。

虽然通过逃逸突变体识别出的大多数残留物都是准确的, 但这种方法的主要注意事项之一是, 逃逸变种的点突变不一定能映射到抗体的分子足迹中, 这是由结构分析。这是由于在一定的残留物上发生突变的能力, 导致了远端到突变残留物位置的构象变化, 类似于构效应。另一个限制是, 这种方法只能用于中和抗体;缺乏体外选择性压力的抗体不会导致变种人逃逸。然而, 这一限制可以通过使用由先前特征中和抗体产生的逃逸变体面板来克服。Tan et al.使用了中和单克隆的转义变体 H7N9 病毒来映射 non-neutralizing 抗体7的表位。
尽管如此, 通过生成逃逸变种来阐明抗体的表位, 为晶体学和低温电子显微镜提供了一种可行的替代方法, 这两者都需要对设备进行广泛的投资。其他的选择是用丙氨酸扫描或肽扫描/截断突变体来确定抗体的最小结合区。在筛选29期间, 丙氨酸扫描诱变可能需要大量的工作来生成大量的变体, 而多肽扫描仅限于线性表位30。本协议中描述的方法不需要特殊的设备或技术, 事实上, 利用现有的体外中和检测来产生感兴趣的抗体的逃逸变种。
用于生成需要多个通道 (例如、茎特异抗体) 的转义变体的协议非常依赖于0通道中抗体的起始浓度。最好是在谨慎的方面犯错, 并在日志中开始的时候要比抗体的最大抑制浓度低一半, 并允许强健的病毒生长。研究人员可以推测, 在低免疫压力的情况下, 高效价病毒培养会在病毒种群中有很大的遗传变异。逃命变形可以被选择为通过逐渐增加抗体集中在以下段落。在病毒生长减少的情况下, 病毒上清的数量可以增加在下一段, 同时保持相同数量的抗体浓度在前一段。
大多数通用流感疫苗的目的是要对 HA 的茎区进行强有力的抗体应答。对茎特异抗体的逃逸变异进行分析, 对于确定流感病毒适应与免疫压力之间的关系具有重要意义。有趣的是, 从茎特异抗体产生的逃逸突变病毒都是在小鼠 LD50研究4中全部衰减的在体内。这些研究为 stalk-based 疫苗接种平台提供了有力的理由。此外, 该协议可用于确定逃逸变种的其他抗病毒化合物, 如小分子抑制剂。最后, 这种方法不限于流感病毒表面糖蛋白, 但也可能更广泛地应用于确定其他病毒糖蛋白的表位。

与其他 RNA 病毒类似, 甲型流感病毒具有易出错的聚合酶, 允许在每一次复制1、2、3中生成多种抗原变体。甲型流感病毒具有惊人的能力来适应和逃避通过抗原漂移的人类免疫反应, 这是通过积累的表面糖蛋白的突变, 导致抗体结合的损失。抗原漂移的病毒表面糖蛋白, HA 和神经氨酸酶 (NA), 需要重新制订和管理的疫苗每年。
分离和生成抗原特异抗体的技术进步产生了大量疫苗诱导的抗体4,5,6,7,8。反过来, 广义中和甲型流感病毒的抗体表位的表征极大地帮助了一些通用流感疫苗候选者的发展9,10,11, 12、13、14。阐明单克隆人的抗原足迹揭示了中和的结构决定因素, 并允许对疫苗设计采取知情的方法。然而, 它既不是现实的, 也不是 cost-effective 的实验室, 以结构表征广泛面板的抗体通过 x 射线晶体学或低温电子显微镜, 以地图表位的病毒抗原15,16,17,18。
x 射线晶体学或低温电子显微镜需要昂贵的设备, 专门的技术和潜在的大量的时间来生成数据。一个替代和更快的方法是利用快速产生不同的病毒群体, 通过容易出错的 rna 相关 rna 聚合酶产生逃逸突变体, 以确定抗体的表位19,20, 21、22、23。产生的逃生变种不需要任何特殊的设备或技术, 并可以执行与传统的实验室试剂和设备。
在这里, 我们描述了一种方法, 允许映射的关键残留物的抗体绑定, 承认流感 HA。

<table border="0" cellpadding="0" cellspacing="0" width="1159">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Falcon 96-well clear flat bottom TC-treated culture microplate with Lid</td>
			<td style="width:197px;">Corning, Inc.</td>
			<td align="right" style="width:119px;">353072</td>
			<td style="width:429px;">Assay plate use for the microneutralization assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Falcon 96-well clear V-bottom plate</td>
			<td style="width:197px;">Corning, Inc.</td>
			<td align="right" style="width:119px;">353263</td>
			<td>Assay plate use for the hemagglutination inhibition assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">1X Minimal Essential Medium (MEM)</td>
			<td style="width:197px;">Gibco</td>
			<td align="right" style="width:119px;">11095080</td>
			<td>Infection medium</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Tosyl phenylalanyl chloromethyl (TPCK)-treated trypsin</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">T8802</td>
			<td>Cleaves immature HA0 to HA1 and HA2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Biotinylated anti-NP primary antibody (IAV)</td>
			<td>EMD Millipore</td>
			<td style="width:119px;">MAB8258B</td>
			<td>An antibody that recognizes the NP protein of influenza A viruses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Biotinylated anti-NP primary antibody (IBV)</td>
			<td style="width:197px;">EMD Millipore</td>
			<td style="width:119px;">MAB8260B-5</td>
			<td>An antibody that recognizes the NP protein of influenza B viruses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Streptavidin-HRP antibody</td>
			<td>EMD Millipore</td>
			<td style="width:119px;">18-152</td>
			<td>This is used as a secondary antibody for the biotinylated anti-NP antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">HRP substrate (SIGMAFAST-OPD)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9187-5SET</td>
			<td>o-phenylenediamine dihydrochloride water soluble substrate for HRP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">96-well V-bottom plate</td>
			<td style="width:197px;">Nunc</td>
			<td align="right" style="width:119px;">249662</td>
			<td>Assay plate used for the hemagglutination assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Chicken red blood cells</td>
			<td>Lampire Biological Laboratories</td>
			<td align="right" style="width:119px;">7201403</td>
			<td>Used to assess the ability of influenza virus to agglutinate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">TRIzol</td>
			<td style="width:197px;">Ambion</td>
			<td align="right" style="width:119px;">15596026</td>
			<td>Extraction of RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Superscript III</td>
			<td style="width:197px;">Invitrogen</td>
			<td align="right" style="width:119px;">12574018</td>
			<td>Reverse transcriptase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Gel Extraction Kit</td>
			<td style="width:197px;">Qiagen</td>
			<td align="right" style="width:119px;">28704</td>
			<td>Isolation of amplified PCR product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Lipofectamine 2000</td>
			<td style="width:197px;">Invitrogen</td>
			<td align="right" style="width:119px;">11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Anti-human IgG (H+L) Alexa Fluor 488</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:119px;">A-11013</td>
			<td>Fluorescent secondary antibody for human antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Anti-mouse IgG (H+L) Alexa Fluor 488</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:119px;">A-11001</td>
			<td>Fluorescent secondary antibody for murine antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">6-well polystyrene microplate</td>
			<td style="width:197px;">Corning, Inc.</td>
			<td align="right" style="width:119px;">353934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">UltraPure Agarose</td>
			<td style="width:197px;">Invitrogen</td>
			<td align="right" style="width:119px;">16500500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Nalgene long term storage Cryo-tubes</td>
			<td style="width:197px;">ThermoFisher Scientific</td>
			<td style="width:119px;">5012-0020</td>
			<td>Freezing of viral culture supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">reassortant A/California/04/09 (H1)</td>
			<td style="width:197px;">Palese Laboratory</td>
			<td style="width:119px;"></td>
			<td>reassortant virus expressing the HA and NA of A/California/04/09 (H1N1) with the internal segments of A/Puerto Rico/8/34 (H1N1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">reassortant A/Shanghai/1/13 (H7)</td>
			<td style="width:197px;">Palese Laboratory</td>
			<td style="width:119px;"></td>
			<td>reassortant virus expressing the HA and NA of A/Shanghai/1/13 (H7N9) with the internal segments of A/Puerto Rico/8/34 (H1N1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Bovine serum albumin solution (35%)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7979</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Qiagen gel extration kit</td>
			<td style="width:197px;">Qiagen</td>
			<td align="right" style="width:119px;">28704</td>
			<td>Silica-membrane-based purification of DNA fragments</td>
		</tr>
	</tbody>
</table>

generation of escape variants of neutralizing influenza virus monoclonal antibodies

流感病毒具有显著的适应和躲避宿主免疫应答的能力。一种方法是通过抗原的变化发生在表面糖蛋白的病毒。转义变种的产生是阐明病毒如何逃脱免疫检测和识别抗体结合所需的关键残留物的有力方法。在这里, 我们描述了一个关于如何通过利用人或小鼠单克隆抗体 (抗体) 的病毒血 (HA) 来产生流感的一个协议。利用我们的技术, 我们以前的特点是需要的关键残留物的抗体结合的头或茎的新的鸟类 H7N9 HA。该协议可以很容易地适应其他病毒系统。逃逸变异的分析是重要的建模抗原漂移, 确定单核苷酸多态性 (snp) 赋予抵抗和病毒健身, 并在设计疫苗和/或治疗。

我们以前使用过这种方法的变化产生的逃逸变种的人和小鼠抗体诱导的季节性流感病毒疫苗, H7N9 疫苗接种, 或顺序 DNA/重组 HA 蛋白疫苗接种4,5 ,6,7。如上所述, 抗体的第一个特点是使用 HI 和 microneutralization 化验, 以便告诉我们哪些特定的协议继续与下一个4,5。抗体 07-5D03, 07-5F01, 07-5G01, 07-4B03, 07-4E02 和07-4D05 被发现有 HI 和中和的活动反对禽 H7N9 病毒 (A/Shanghai/1/2013) (表 1), 因此使用了协议 1 (步骤 2.1)。对于缺少 HI 活动的抗体, 如41-5E04、045-051310-2B06、042-100809-2F04 和 S6-B01 (表 1), 协议 2 (步骤 2.2) 用于生成转义变体。逃逸突变体映射显示, 许多抗体识别在病毒 HA4,5 (图 4) 的不同位置的关键残留物。虽然大多数高阳性抗体有逃脱突变残留附近的前报告抗原点的 H7 HA, 高阴性抗体产生逃逸突变体与点突变的茎区4,5.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>抗体</td>
			<td>您好活动</td>
			<td>NEUT 活动</td>
		</tr>
		<tr>
			<td>07-5D03</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>07-5F01</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>07-5G01</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>07-4B03</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>07-4E02</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>07-4D05</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>41-5E04</td>
			<td>-</td>
			<td>+</td>
		</tr>
		<tr>
			<td>045-051310-2B06</td>
			<td>-</td>
			<td>+</td>
		</tr>
		<tr>
			<td>042-100809-2F04</td>
			<td>-</td>
			<td>+</td>
		</tr>
		<tr>
			<td>S6-B01</td>
			<td>-</td>
			<td>+</td>
		</tr>
	</tbody>
</table>
表 1:抗体 HI 和中和活动表.十 H7-specific 抗体从个人接种的实验性 H7N9 疫苗中分离出不同的体外抗病毒活性5。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">正向底漆 (5 "到 3")</td>
			<td colspan="4">反向底漆 (5 "到 3")</td>
			<td colspan="2">Thermocylcer 条件</td>
		</tr>
		<tr>
			<td>IAV</td>
			<td colspan="4">TATTCGTCTCAGGGAGCAAAAGCAGGGG</td>
			<td colspan="4">ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT</td>
			<td colspan="2">42 c 为 60 min, 94 c 为 2 min/5 周期 94 c 为二十年代, 50 c 为三十年代和 68 c 为3分钟三十年代, 其次40周期 94 c 为二十年代, 58 c 为三十年代和 68 c 为 3 min 三十年代以最后的延长时间在 68 c 为10分钟</td>
		</tr>
		<tr>
			<td>ibv</td>
			<td colspan="4">GGGGGGAGCAGAAGCAGAGC</td>
			<td colspan="4">CCGGGTTATTAGTAGTAACAAGAGC</td>
			<td colspan="2">45 c 为 60 min, 55 c 为 30 min, 94 c 为 2 min/5 周期 94 c 为二十年代, 40 c 为三十年代和 68 c 为3分钟三十年代, 其次40个周期 94 c 为二十年代, 58 c 三十年代和 68 c 为 3 min 三十年代以最后的延长时间在 68 c 为10分钟</td>
		</tr>
	</tbody>
</table>
表 2: 通用流感病毒引物.底漆对放大的 HA 部分的流感 A27和 B28病毒及其各自的 thermocycler 条件。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56067/56067fig1.jpg"> 
图 1: HI 化验.(a) 用于设置 HI 检测的示意图, 用于测试两个鼠标 H1-specific 抗体 7B2 (特定于头) 和 6F12 (特定于茎) 的活动, 使用96井 V 底板, 和 (B) 一个 HI 检测结果的示例23。<a href="//ecsource.jove.com/files/ftp_upload/56067/56067fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56067/56067fig2.jpg"> 
图 2: Microneutralization 检测.用于设置 microneutralization 检测的示意图, 用于测试两个人类抗体 4D055和 CR911417的活动。<a href="//ecsource.jove.com/files/ftp_upload/56067/56067fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56067/56067fig3.jpg"> 
图 3: 转义变种的生成.所提出的方法将取决于抗体所展示的 HI 和 microneutralization 活性。对 (A) 中和 hi 阳性抗体的逃逸突变体的产生可能需要在卵中有一个通道, 而 (B) 中和高阴性抗体可能涉及多个通道, 抗体量增加细胞组织培养。<a href="//ecsource.jove.com/files/ftp_upload/56067/56067fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56067/56067fig4.jpg"> 
图 4: 以转义变种变种生成的新型禽 H7N9的表位图的示例。疫苗诱导的抗体分离的个人接种的候选 H7N9 流感疫苗被用来产生逃逸变种。用红色表示的每一个残留物代表了一个单克隆抗体有效结合所需的关键氨基酸的位置。数据被改编自 Dunand-亨利et al., 20154。<a href="//ecsource.jove.com/files/ftp_upload/56067/56067fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies at JoVE.com

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

我们描述一种方法, 我们确定的关键残留物所需的结合的人或小鼠单克隆抗体的目标病毒血甲型流感病毒。该协议可以适应其他病毒表面糖蛋白及其相应的中和抗体。

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 ...

generation-escape-variants-neutralizing-influenza-virus-monoclonal

Research

JoVE Journal

Immunology and Infection

11.6K 次观看.  Icahn School of Medicine at Mount Sinai. 我们描述一种方法, 我们确定的关键残留物所需的结合的人或小鼠单克隆抗体的目标病毒血甲型流感病毒。该协议可以适应其他病毒表面糖蛋白及其相应的中和抗体。

视频: Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

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 ...

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, ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

鼠单克隆抗体的产生杂交瘤技术

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

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 ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...