研究方案通过当地道德审查委员会 (www.EKNZ.ch) 批准, 并获得了所有与会者的书面知情同意。
1. 血清采集
<ol><li>在感兴趣的时间点从人体收集血清样本。在这项研究中, 我们收集的血清在0天 (流感疫苗接种时间), + 7, + 30, + 60, 和 +180 后接种。</li>
	<li>为了获得血清, 离心的样品管在 1200 x g 为10分钟室温 (20-25 ° c)。 注: 非离心血液样品应贮存在4° c, 且不超过24小时。</li>
	<li>将血清分到不同的管中 (冷冻瓶), 并在-80 ° c 冷冻直至使用。</li>
	<li>执行随后的化验批次-明智的, 包括一个人的所有时间点, 以减少在病人的变异性。</li>
</ol>2. 抗原的制备
警告: 使用五种不同的抗原 (请参见材料表)。在生物安全2级 (BSL-2) 实验室制备抗原。
<ol><li>根据制造商的指示, 重建一个冻干流感抗原安瓿的总内容与1.0 毫升蒸馏水, 并允许溶解抗原在室温下至少站立5分钟, 然后再继续。</li>
	<li>将抗原溶液分至1.5 毫升管, 并在-80 ° c 冷冻, 直至进一步使用。</li>
</ol>3. 霍乱滤液的制备
注: 霍乱滤液根据 WHO 协议12被用作受体破坏酶 (RDE)。这将消除血清中的天然抑制剂, 这将干扰化验17。
<ol><li>根据制造商的说明重组冻干 RDE。</li>
	<li>将 RDE 溶液储存在4° c 的15毫升管中直到进一步使用。</li>
</ol>4. HA 测定
注意: 为了确保高含量的样品在几个板块之间是可比较的, 每个盘子必须使用相同数量的病毒颗粒。ha 检测 (也称为 ha 滴定) 是用来量化的病毒粒子的血凝必要的, 并记录在 ha 单位。血凝的 "单位" 是一个操作单位, 依赖于用于 HA 滴定的方法, 而不是测量绝对数量的病毒。因此, HA 单位被定义为凝集标准红细胞悬浮液的等量量所需的病毒数量。根据 WHO 的标准量, 用于高含量的检测是每25µL 4 公顷的单位。有关 HA 测定的原理的说明, 请参见图 1。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55833/55833fig1.jpg" style="font-size: 14px;"> 图 1: 血凝和血凝抑制的原理.在没有病毒和抗体 (左栏) 的阴性控制情况下, 没有血凝发生, 只有在流感病毒 (中柱) 存在的红细胞 hemagglutinate。然而, 当流感病毒的血被特定病毒抗体阻断时, 就不会发生血凝 (右栏)。<a href="//cloudflare.jove.com/files/ftp_upload/55833/55833fig1large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>
注: 所使用的红细胞取决于检测中的流感病毒类型 (表 1)。此外, 对于各种类型的96井微滴板, 孵化时间以及非粘结细胞的外观不同 (表 2)。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>流感抗原</td>
			<td>A/California/7/09 (H1N1)</td>
			<td colspan="2">A/Switzerland/9715293/2013 (H3N2)</td>
			<td>A/Texas/50/2012 (H3N2)</td>
			<td>B/Brisbane/60/08</td>
			<td colspan="2">B/Massachusetts/02/2012</td>
		</tr>
<tr>
<td>红细胞种类</td>
			<td>鸡</td>
			<td colspan="2">豚鼠</td>
			<td>豚鼠</td>
			<td>土耳其</td>
			<td colspan="2">土耳其</td>
		</tr>
</tbody></table>表 1: 流感抗原及相应种类的红细胞。根据制造商的说明 (NIBSC)。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>红细胞种类</td>
			<td>鸡</td>
			<td>土耳其</td>
			<td>豚鼠</td>
			<td>人型 O</td>
		</tr>
<tr>
<td>红细胞浓度 (v/v)</td>
			<td>0.75%</td>
			<td>0.75%</td>
			<td>1%</td>
			<td>1%</td>
		</tr>
<tr>
<td>孔板类型</td>
			<td>V 底</td>
			<td>V 底</td>
			<td>U 底</td>
			<td>U 底</td>
		</tr>
<tr>
<td>孵化时间, RT</td>
			<td>30分钟</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>1小时</td>
		</tr>
<tr>
<td>非粘结细胞的外观</td>
			<td>按钮*</td>
			<td>按钮*</td>
			<td>光环</td>
			<td>光环</td>
		</tr>
</tbody></table>表 2: 不同种类的红细胞的测定条件.根据世卫组织的议定书。(* 倾斜时流动)。
<ol><li>
红细胞悬浮液的制备
	<ol>
<li>稀释红细胞悬浮物 (10%, v/v; 除了人类类型 O) (见材料表) 与磷酸盐缓冲盐水 (PBS), 使适当浓度的鸟类和哺乳动物红细胞的0.75% 和 1%, 分别。</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55833/55833fig2.jpg" style="font-size: 14px;"> 图 2: HA 检测的板设计.HA 滴定是重复执行的。控制行中没有添加抗原。还请参见图 4确定最佳抗原浓度。<a href="//cloudflare.jove.com/files/ftp_upload/55833/55833fig2large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>
96井微滴板的制备 注意: 有关板设计的概述, 请参见图 2 。<ol>
<li>使用多通道吸管 (图 2), 在96阱微滴板的每行使用1至12的µL 中添加25的 PBS。使用 V 型微滴板时, 与禽红细胞, 如鸡肉和土耳其工作。使用 U 型微滴板时, 与哺乳动物红细胞, 如豚鼠和人类类型 O (表 2) 工作。</li>
		<li>在抗原行的第一个井中加入25µL 流感抗原, 这是以重复的方式排列的。控制行中不添加抗原。控制行不应显示血凝效果并充当负控制 (图 2)。</li>
		<li>通过使用多通道吸管将25µL 从抗原行的第一口井转移到连续井, 进行连续2倍稀释。每稀释一步由移上下轻轻10次。</li>
		<li>丢弃最后一口井的最后25µL。</li>
		<li>使用多通道吸管将 PBS 的25µL 添加到1到12的每一个行中, 以达到每井50µL 的总体积。</li>
		<li>加入50µL 的红细胞悬浮液, 每一个使用多通道吸管。</li>
		<li>小心地轻敲盘子10次在所有四边混合。</li>
		<li>用盖子盖住盘子, 在室温下孵育适当的时间, 这取决于所使用的红细胞种类 (参见表 2)。在孵化时不要移动盘子。</li>
	</ol>
</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55833/55833fig3.jpg" style="font-size: 14px;"> 图 3: 鸟类和哺乳动物红细胞的凝集模式.在与禽红细胞工作时, 使用 V 型微滴板。读数是在倾斜的板块位置, 非粘结红细胞开始运行下来形成一个撕裂状的形状。U 形孔板使用时, 与哺乳动物红细胞。然后在非倾斜位置进行读数, 非粘结红细胞形成一个小光环。<a href="//cloudflare.jove.com/files/ftp_upload/55833/55833fig3large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="3"><li>
阅读板 注: 与哺乳动物红细胞相比, 由于不同形状的微滴度井 (图 3), 在使用禽红细胞时, 读数略有不同。<ol>
<li>禽红细胞的读数<ol>
<li>倾斜板90°二十五年代。 注: 倾斜板对禽型的分化至关重要, 因为所有三种不同类型的凝集模式 (完全粘结、部分粘结和非粘结) 在不倾斜时显示为按钮。</li>
			<li>立即标记结果, 而该板仍处于倾斜位置, 在96井板的打印方案。禽红细胞的凝集模式如图 3所示。</li>
		</ol>
</li>
		<li>哺乳动物红细胞的读数<ol>
<li>将结果标记在96井板的打印方案上, 而不倾斜板 (工作台的水平位置)。 注: 当血凝发生时, 粘结细胞不会沉淀到底部, 而非粘结细胞则会出现在井底的光环中。部分粘结的细胞的晕强度较小, 直径较大 (图 3)。</li>
		</ol>
</li>
		<li>4公顷单位的测定。 注: HA 滴定终点是完全血凝发生的最后一处井。这井包含1公顷的病毒单位。由于抗原的2倍稀释, 在 ha 滴定终点之前的两口井是包含4公顷病毒的井 (图 4)。</li>
	</ol>
</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55833/55833fig4.jpg" style="font-size: 14px;"> 图 4: ha 滴定与禽红细胞的读数, 以确定 4 ha 单位的效价.用血凝试验 (抗原滴定法) 测定血凝所需的最佳抗原量。最后一井, 完全血凝发生是 ha 滴定终点和包含1公顷单位。由于抗原的2倍稀释, 在 ha 滴定终点之前有两口井, 其滴度对应于4公顷的单位。<a href="//cloudflare.jove.com/files/ftp_upload/55833/55833fig4large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>
5. HI 检测
注意: 该协议的工作流程已经过优化, 可以同时使用 PCR 管条纹和热循环更有效地处理多个样品。
<ol><li>
血清标本的制备 注: 在 BSL-2 实验室制备血清样品。<ol>
<li>在室温下解冻每个人的每个时间点的冰冻血清样本 (见步骤 1.2)。</li>
		<li>将每个解冻的血清样本的分10µL 添加到 PCR 管条 (一条带10管) 的试管中。 注: 使用 PCR 管条的最大优点是多通道吸管可用于以下步骤的 HI 测定;这节省了大量的时间, 当测试大量的血清样本和执行重复的措施相同的样本抗体滴对不同的病毒株。</li>
		<li>将 aliquoted 血清样本储存在-80 ° c 的 PCR 管条中直到使用。</li>
		<li>在进行 HI 化验前的一天, 在室温下解冻血清样品等分。</li>
		<li>在一个空的 PCR 管中加入10µL 的适当抗血清。 注意: 作为一个积极的控制, 抗血清对特定的病毒必须匹配使用的病毒。正向控制允许在多个板上对板的性能进行标准化。</li>
		<li>使用多通道吸管将30µL 的霍乱滤液溶液添加到每个血清分和抗血清 (3 卷霍乱滤液到1的血清) 中。</li>
		<li>将 pcr 管放在 pcr 的96井机架上, 或者是一个空的尖盒和 5 s 的涡流。</li>
		<li>用热循环在37° c 的夜间孵育样品。</li>
		<li>在56° c 孵育样品30分钟, 用热循环将霍乱滤液灭活。 注: 根据热循环, 此步骤可编程以进一步自动化过程。</li>
		<li>将 pcr 管放在 pcr 的96井机架上, 或者是一个空的尖盒和 5 s 的涡流。</li>
		<li>将样品储存在4° c 的冰箱中, 直到用于 HI 化验。</li>
	</ol>
</li>
</ol><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55833/55833fig5v2.jpg" style="font-size: 14px;"> 图 5: 板的设计和工作流程的 HI 检测.两个人的五时间点可以在一个板块上测量。高滴度范围从8到1024。对所用抗原的抗血清作阳性对照, 并进行反滴定检查抗原稀释是否等于 4 HA 单位。血清样本的序列稀释显示为2个人疫苗接受者。 <a href="//cloudflare.jove.com/files/ftp_upload/55833/55833fig5v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>
HI 检测 注: 为高分析的原则的例证参见图 1。根据病毒, 不同种类的红细胞用于检测 (表 1)。不同种类的红细胞用于各种类型的 96-井板, 孵化时间以及非粘结细胞的外观不同 (表 2)。对于 HI 化验, 4 公顷的病毒或抗原的单位被添加到2倍稀释系列的样品。<ol>
<li>
抗原溶液的制备
		<ol>
<li>根据所使用的96井板的数量计算所需抗原溶液的体积 (25 µL 抗原每 96 = 2400 µL 抗原每 96-井板; 增加100µL 每盘由于多通道吸管的使用水库; 共2.5 毫升的 antigen 每盘)。 注: 例如, 如果测量100血清样本, 则需要10板 (每盘10样品): 2.5 毫升 x 10 = 25 毫升的抗原溶液总共需要。</li>
			<li>使用 PBS 为计算出的容积准备适当稀释 4 HA 单位。 注: 4 公顷单位是确定的 ha 化验。对于适当的抗原量, 除以与 4 HA 单位对应的滴度计算的体积。例如, 4 HA 单位对应于稀释 1/64, 并且我们需要1.5万µL 抗原解答是需要的: 1.5万/64 = 234.4 µL 溶解冻干流感抗原被增加。</li>
		</ol>
</li>
		<li>
红细胞悬浮液的制备
		<ol>
<li>根据所使用的96井微滴度板的数量计算红细胞悬浮液的体积 (50 µL 红细胞悬架每井 x 96 = 4800 µL 红细胞悬浮每 96-井板; 添加200µL 每板额外由于使用了一个水库为多通道吸管).</li>
			<li>稀释红细胞悬浮液 (通常为 10%, v/v; 除了人型 O) 与 PBS, 使适当的浓度为鸟类和哺乳动物红细胞的0.75% 和1% 分别。</li>
		</ol>
</li>
		<li>
96井微滴板的制备
		<ol>
<li>标签96井微滴板 (样品 ID, 阳性对照, 和背部滴定)。请仔细检查图 5中的板方向。</li>
			<li>除了 "后滴定" 行的第一个井 (图 5, 12th行), 使用多通道吸管将 PBS 的25µL 添加到每个井。 注: 采用后滴定法检查所使用的抗原稀释是否等于4公顷的单位。如果在 "后滴定" 排的前三井中发生血凝, 而在第四井则不存在, 则表明 4 HA 单位的抗原效价。</li>
			<li>在 "后滴定" 行 (12th行) 的第一个井中添加50µL 的准备抗原溶液 (在5.2.1 中描述)。</li>
			<li>使用多通道吸管, 将 RDE 处理过的血清样品的25µL 添加到每片1至10排的第一口井。</li>
			<li>将适当抗血清的25µL 添加到 11th行的第一个井中作为阳性对照。</li>
			<li>通过使用多通道吸管将25µL 从每行 (1-12) 的第一口井转移到连续井, 执行串行2倍稀释。混合由移上下 10-15 倍为每个稀释步骤。同样的提示可以用于每一个稀释步骤的每个样品。</li>
			<li>丢弃最后一口井的最后25µL。</li>
			<li>增加25µL 的抗原溶液, 使用多通道吸管, 每行1至 11 (血清样本和抗血清)。如果他们不接触水井, 也可以使用同样的技巧。</li>
			<li>在 "后滴定" 行 (12th行) 的每个井中添加25µL 的 PBS 而不是抗原。</li>
			<li>小心地轻敲盘子10次在所有四边混合。</li>
			<li>用盖子盖住盘子, 在室温下孵育30分钟。在孵化时不要移动盘子。</li>
			<li>每井加50µL 的红细胞悬浮液。</li>
			<li>小心地轻敲盘子10次在所有4边混合。</li>
			<li>用盖子盖住盘子, 在室温下孵育适当的时间, 这取决于所使用的红细胞种类 (参见表 2)。在孵化时不要移动盘子。</li>
		</ol>
</li>
		<li>
阅读板 注: HI 滴度是最后稀释 (抗) 血清的倒数, 完全抑制血凝。重要的是要考虑到, RDE 处理的血清已经稀释 1:4, 并在连续稀释步骤后, 在第一口井的血清稀释 1:8, 这相当于一个 HI 效价8。<ol>
<li>禽红细胞的读数<ol>
<li>倾斜板90°二十五年代。 注: 倾斜板对禽型的分化至关重要, 因为所有三种不同类型的凝集模式 (完全粘结、部分粘结和非粘结) 在不倾斜时显示为按钮。</li>
				<li>立即标记结果, 而该板仍处于倾斜位置, 在96井板的打印方案。禽红细胞的凝集模式如图 3所示。</li>
			</ol>
</li>
			<li>哺乳动物红细胞的读数<ol>
<li>在不倾斜板的情况下, 将结果标记为96井板的打印方案。 注: 当血凝发生时, 粘结细胞不会沉淀下来, 而非粘结细胞则会出现在井底的光环中。部分粘结的细胞的晕强度较小, 直径较大 (图 3)。</li>
				<li>确定每个示例的 HI 并将其传输到基于计算机的表 (图 6)</li>
				<li>注: 部分粘结井被确定为较低滴度。例如, 如果一个血清样本完全抑制了凝血多达第四井 (1:64 稀释) 和 5th井 (1:128 稀释) 是部分粘结, 然后高滴度是设置到较低的效价64的最终分析 (图 6,4th行)。</li>
			</ol>
</li>
		</ol>
</li>
	</ol>
</li>
</ol><img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55833/55833fig7.jpg"> 图 6: 使用禽红细胞的 HI 检测的读数.通过 HI 试验测定了接种前后的流感特异抗体应答。在这个例子中, 人一有高滴比人二。两人在接种疫苗后均表现出抗体应答;180天在疫苗接种以后两个人的抗体滴再减少。<a href="//cloudflare.jove.com/files/ftp_upload/55833/55833fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+seasonal+influenza+with+vaccines.+Recommendations+of+the+Advisory+Committee+on+Immunization+Practices--United+States,+2013-2014.">Prevention and control of seasonal influenza with vaccines. Recommendations of the Advisory Committee on Immunization Practices--United States, 2013-2014.</a> MMWR Recomm Rep. 62, 1-43 (2013).</li><li>Dominguez-Cherit, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critically+Ill+patients+with+2009+influenza+A(H1N1)+in+Mexico.">Critically Ill patients with 2009 influenza A(H1N1) in Mexico.</a> JAMA. 302 (17), 1880-1887 (2009).</li><li>Fox, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandemic+influenza+(H1N1):+impact+on+lung+transplant+recipients+and+candidates.">Pandemic influenza (H1N1): impact on lung transplant recipients and candidates.</a> J Heart Lung Transplant. 29 (9), 1034-1038 (2010).</li><li>Piercy, J., Miles, A., Krankheiten, S. B. f. G. S. V., Values, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Economic Impact of Influenza in Switzerland: Interpandemic Situation. , (2003).</li><li>Barclay, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+network+assortativity+of+influenza+vaccination+at+a+high+school:+implications+for+outbreak+risk+and+herd+immunity.">Positive network assortativity of influenza vaccination at a high school: implications for outbreak risk and herd immunity.</a> PLoS One. 9 (2), 87042 (2014).</li><li>Baluch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomized+controlled+trial+of+high-dose+intradermal+versus+standard-dose+intramuscular+influenza+vaccine+in+organ+transplant+recipients.">Randomized controlled trial of high-dose intradermal versus standard-dose intramuscular influenza vaccine in organ transplant recipients.</a> Am J Transplant. 13 (4), 1026-1033 (2013).</li><li>Haralambieva, I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Immunosenescence+on+Humoral+Immune+Response+Variation+after+Influenza+A/H1N1+Vaccination+in+Older+Subjects.">The Impact of Immunosenescence on Humoral Immune Response Variation after Influenza A/H1N1 Vaccination in Older Subjects.</a> PLoS One. 10 (3), 0122282 (2015).</li><li>Egli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccine+adjuvants--understanding+molecular+mechanisms+to+improve+vaccines.">Vaccine adjuvants--understanding molecular mechanisms to improve vaccines.</a> Swiss Med Wkly. 144, 13940 (2014).</li><li>O'Shea, D., Widmer, L. A., Stelling, J., Egli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+face+of+vaccination+in+immunocompromised+hosts.">Changing face of vaccination in immunocompromised hosts.</a> Curr Infect Dis Rep. 16 (9), 420 (2014).</li><li>Meulemans, G., Carlier, M. C., Gonze, M., Petit, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+hemagglutination-inhibition,+agar+gel+precipitin,+and+enzyme-linked+immunosorbent+assay+for+measuring+antibodies+against+influenza+viruses+in+chickens.">Comparison of hemagglutination-inhibition, agar gel precipitin, and enzyme-linked immunosorbent assay for measuring antibodies against influenza viruses in chickens.</a> Avian Dis. 31 (3), 560-563 (1987).</li><li>Martins, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+internal+controls+for+the+Luminex+instrument+as+part+of+a+multiplex+seven-analyte+viral+respiratory+antibody+profile.">Development of internal controls for the Luminex instrument as part of a multiplex seven-analyte viral respiratory antibody profile.</a> Clin Diagn Lab Immunol. 9 (1), 41-45 (2002).</li><li>Webster, R., Cox, N., St&#246;hr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+Animal+Influenza+Manual.">WHO Animal Influenza Manual.</a> WHO/CDS/CSR/NCS. 2002.5, 1-99 (2002).</li><li>Mittelholzer, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cell+lines+used+in+a+micro+neutralization+test+for+measuring+influenza-neutralizing+antibodies.">Human cell lines used in a micro neutralization test for measuring influenza-neutralizing antibodies.</a> Scand J Immunol. 63 (4), 257-263 (2006).</li><li>Hamilton, R. S., Gravell, M., Major, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+antibody+titers+determined+by+hemagglutination+inhibition+and+enzyme+immunoassay+for+JC+virus+and+BK+virus.">Comparison of antibody titers determined by hemagglutination inhibition and enzyme immunoassay for JC virus and BK virus.</a> J Clin Microbiol. 38 (1), 105-109 (2000).</li><li>Kumakura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+hemagglutination+inhibition+assay+and+enzyme+immunoassay+for+determination+of+mumps+and+rubella+immune+status+in+health+care+personnel.">Comparison of hemagglutination inhibition assay and enzyme immunoassay for determination of mumps and rubella immune status in health care personnel.</a> J Clin Lab Anal. 27 (5), 418-421 (2013).</li><li>Ogundiji, O. T., Okonko, I. O., Adu, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+measles+hemagglutination+inhibiting+antibody+levels+among+school+children+in+Ibadan,+Nigeria.">Determination of measles hemagglutination inhibiting antibody levels among school children in Ibadan, Nigeria.</a> J Immunoassay Immunochem. 34 (2), 208-217 (2013).</li><li>Cwach, K. T., Sandbulte, H. R., Klonoski, J. M., Huber, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+murine+innate+serum+inhibitors+toward+interference+within+influenza+virus+immune+assay.">Contribution of murine innate serum inhibitors toward interference within influenza virus immune assay.</a> Influenza Other Respir Viruses. 6 (2), 127-135 (2012).</li><li>Lee, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+mimicry+by+antibody+F045-092+facilitates+universal+binding+to+the+H3+subtype+of+influenza+virus.">Receptor mimicry by antibody F045-092 facilitates universal binding to the H3 subtype of influenza virus.</a> Nat Commun. 5, 3614 (2014).</li><li>Blumel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+prevalence+of+cross-reactive+antibodies+against+influenza+A(H3N2)+variant+virus,+Germany,+2003+to+2010.">Age-related prevalence of cross-reactive antibodies against influenza A(H3N2) variant virus, Germany, 2003 to 2010.</a> Euro Surveill. 20 (32), 16-24 (2015).</li><li>Reber, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+Influenza+Vaccination+of+Children+Induces+Humoral+and+Cell-Mediated+Immunity+Beyond+the+Current+Season:+Cross-reactivity+with+Past+and+Future+Strains.">Seasonal Influenza Vaccination of Children Induces Humoral and Cell-Mediated Immunity Beyond the Current Season: Cross-reactivity with Past and Future Strains.</a> J Infect Dis. , (2016).</li></ol>

a.e 得到了 "SNSF Ambizione 评分" 计划 (PZ00P3_154709)、"Forschungsfond、Förderung strategischer Projekte" 巴塞尔大学、Stiftungsinfektionskrankheiten 巴塞尔和 Bangeter Rhyner 基金会的研究资助。力得到奥地利格拉茨技术大学的资助。. 承认, 在系统生物学计划 (9th调用) 的 SystemsX.ch 倡议的 iPhD 奖学金的支持。

对疫苗接种前后流感病毒特异性抗体滴的定量是一种重要的研究手段。基于保护病毒感染的替代措施, 如 seroprotection (> 1:40) 或血清 (4 倍效价增加), 疫苗接种策略可以优化9。使用所提供的协议可以确定: (i) 特定病毒的血凝潜能, 和 (ii) 抗体滴的病毒的利益。
修改和故障排除:
此协议基于 WHO 标准12。我们用 PCR...

感染流感病毒与相当大的发病率, 死亡率和高医疗费用相关的1,2,3,4。特别是, 老年人、新生儿、孕妇和慢性病患者面临更严重的临床后果。因此, 预防循环流感病毒株的接种是降低高危人群疾病负担的主要措施。疫苗接种后个体免疫应答的增加,例如, 高于保护性阈值的流感特异性抗体, 降低了个体感染的风险, 并在总体上减少了病毒在人群中传播的可能性5. 详细了解不同人群及各年龄组的疫苗诱导体液免疫应答是回答重要临床问题的关键因素6、7、8,9, 例如: 为什么有些老年患者尽管曾接种过疫苗, 但仍有感染？什么是 "好" 和 "足够" 疫苗诱导的保护？疫苗应多久免疫患者才能达到保护性滴？什么是最有效的剂量？新佐剂对后接种抗体滴的影响是什么？对疫苗特异抗体的测定可以帮助回答这些重要问题, 提高疫苗接种效果。
病毒特异性抗体滴的定量可以用各种免疫学方法进行。这包括固相10或基于磁珠的 ELISA11化验、HI 化验12和中和分析13。以 ELISA 为基础的方法可以筛选相对大量的血清标本对各种抗原。另外, 可以分别探讨病原体特异免疫球蛋白 (Ig) 和 IgG。虽然抗原的特征,如例如, 线性氨基酸序列或类似病毒的微粒可能影响抗体的结合, 潜在的表位的光谱是非常宽广的并且不提供信息关于是否抗体响应具有功能相关性。
与此相反, 中和试验确定了抗体在功能上抑制细胞感染的潜能, 从而反映了中和电位。然而, 这种方法是非常劳动密集型, 需要培养特定的细胞系和活病毒, 因此, 它是费时, 昂贵, 并要求特殊设备。
本文介绍了一个循序渐进的世界卫生组织 (WHO) 为基础的 HI 协议12 , 以量化流感特异性抗体滴。血凝是某些病毒导致红细胞凝集的一种特征性效应。这种效果与患者血清的抑制, 使抑制抗体浓度的测量, 这反映了中和效应。
我们修改了 WHO 协议的工作流程, 以便在同一时间更有效地处理多个样本, 从而减少所需的时间。第一个协议描述了特定流感抗原的凝集电位的测定。这样做, 确定了正确的流感抗原浓度的第二个协议。这部分应重复每一个新的病毒抗原, 以及每一批血液。
第二个协议描述了流感特异性抗体滴的测定。针对流感病毒和人血清样本的研究, 提出的协议进行了优化但是, 它也可以用于小鼠血清样本或细胞培养清的刺激免疫细胞,例如, 特定病毒 B 细胞。结果可以确定为绝对测量滴。在许多疫苗研究中, 每个特定种群都显示了几何平均滴和95% 置信区间。对于解释, seroprotection 或血清经常被用来描述人口对某种病毒的易感性。Seroprotection 被定义为≥1:40 的效价, 血清为超过4倍的效价增加与 seroprotective 滴在两个时间点之间的成就 (最常见的预接种和30天后接种使用)。
这两种协议都很容易使用, 而且可以适应范围广泛的研究问题。特别是, 他们可以用来确定可靠和快速的抗体滴对其他各种病毒的血凝能力, 如麻疹, polyomaviruses, 腮腺炎, 或风疹14,15,16.

<table><tbody><tr><td>25 ml Disposable Multichannel Pipette Reservoirs</td><td>Integra</td><td>4312</td><td></td></tr><tr><td>8-well PCR tubes</td><td>Brand GMBH</td><td>781332</td><td>For serum aliquots</td></tr><tr><td>96-well microtiter plate, U-shaped</td><td>TPP</td><td>92097</td><td>For HI assay when using mammalian RBCs</td></tr><tr><td>96-well microtiter plate, V-shaped</td><td>Corning Costar</td><td>3897</td><td>For HI assay when using avian RBCs</td></tr><tr><td>Aqua ad iniect. Steril</td><td>Bichsel AG</td><td>1000004</td><td>For preparing influenza antigen and cholera filtrate solutions</td></tr><tr><td>Chicken RBC (10%)</td><td>Cedarlane</td><td>CLC8800</td><td>10% suspension of chicken red blood cells in Alsever&#039;s solution</td></tr><tr><td>Cholera filtrate</td><td>Sigma-Aldrich</td><td>C8772</td><td>Used as receptor destroying enzyme (RDE)</td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td>For diluting the serum samples, RBCs and antigens</td></tr><tr><td>Eppendorf Multichannel pipette, 12-channel, 10-100 &amp;micro;l</td><td>Sigma-Aldrich</td><td>Z683949</td><td></td></tr><tr><td>Eppendorf Multichannel pipette, 8-channel, 10-100 &amp;micro;l</td><td>Sigma-Aldrich</td><td>Z683930</td><td></td></tr><tr><td>Guinea Pig RBC (10%)</td><td>Cedarlane</td><td>CLC1800</td><td>10% suspension of guinea pig red blood cells in Alsever&#039;s solution</td></tr><tr><td>Influenza Anti-A/California/7/09 HA serum&amp;nbsp;</td><td>NIBSC</td><td>14/134&amp;nbsp;</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-A/Switzerland/9715293/2013-like HA serum&amp;nbsp;</td><td>NIBSC</td><td>14/272</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-A/Texas/50/2012-Like HA Serum&amp;nbsp;</td><td>NIBSC</td><td>13/178</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-B/Brisbane/60/2008-HA serum&amp;nbsp;</td><td>NIBSC</td><td>13/254&amp;nbsp;</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-B/Massachusetts/02/2012 HA serum&amp;nbsp;</td><td>NIBSC</td><td>13/182</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza antigen A/California/7/09 (H1N1)(NYMC-X181)&amp;nbsp;</td><td>NIBSC</td><td>12/168</td><td>Inactivated, partially purified A/California/7/09 (H1N1)(NYMC-X181)&amp;nbsp; virus (ca. 46&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen A/Switzerland/9715293/2013 (NIB88)</td><td>NIBSC</td><td>14/254</td><td>Inactivated, partially purified A/Switzerland/9715293/2013 (NIB88) virus (ca. 55&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen A/Texas/50/2012 (H3N2)(NYMCX-223)</td><td>NIBSC</td><td>13/112</td><td>Inactivated, partially purified A/Texas/50/2012 (H3N2)(NYMCX-223) virus (ca. 74&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen B/Brisbane/60/2008</td><td>NIBSC</td><td>13/234</td><td>Inactivated, partially purified B/Brisbane/60/2008 virus (ca. 42&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen B/Massachusetts/02/2012</td><td>NIBSC</td><td>13/134</td><td>Inactivated, partially purified B/Massachusetts/02/2012 virus (ca. 35&amp;micro;gHA/ml)</td></tr><tr><td>Serum-Tubes</td><td>S-Monovette, Sardstedt</td><td>01.1601.100</td><td>For serum extraction with clotting activator</td></tr><tr><td>Single Donor Human RBC, Type 0</td><td>Innovative Research</td><td>IPLA-WB3&amp;nbsp;</td><td>Suspension of single donor human red blood cells in Alsever&#039;s solution (ca. 26%)</td></tr><tr><td>Turkey RBC (10%)</td><td>Cedarlane</td><td>CLC1180</td><td>10% suspension of turkey red blood cells in Alsever&#039;s solution</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td></td><td></td></tr></tbody></table>

an optimized hemagglutination inhibition (hi) assay to quantify influenza-specific antibody titers

抗体滴是常用的替代标志, 用于血清保护流感和其他病原体。对抗体产生前和后接种的详细知识需要了解疫苗诱导免疫。本文介绍了一个可靠的点对点协议来确定流感特异抗体滴。第一个协议描述了一种方法来指定血凝所需的抗原量, 它标准化了第二个协议中随后使用的浓度 (血凝试验, HA 测定)。第二个协议描述了对不同病毒株的流感特异性抗体滴的定量化, 使用人血清或细胞培养清 (血凝抑制法, HI 测定) 的序列稀释。
作为一个应用实例, 我们展示了一个健康的队列的抗体应答, 它接受了三价灭活流感疫苗。此外, 还说明了不同流感病毒之间的交叉反应性, 并解释了使用不同类型的动物红细胞 (红细胞) 减少交叉反应性的方法。讨论突出了所提出的化验的优缺点, 以及如何确定流感特异抗体滴可以提高对疫苗相关免疫的认识。

预防流感 H3N2 前和后接种诱导抗体应答 26健康志愿者接受了疫苗诱导的抗体应答, 他们在2014之前收到了含有流感 A/H1N1/California/2009、A/H3N2/Texas/2012 和 B/Massachusetts/02/2012 的灭活三价亚基流感疫苗。2015流感季节。图 6显示了2疫苗接收者的代表性示例。有趣的是, 在那个特定的流感季节, A/H3N2/Texas/2012 没有循环, 相反, 这个季节包括了一些不?...

Watch this Scientific Journal Video about An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers at JoVE.com

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

本协议描述了如何进行血凝抑制试验, 以量化流感疫苗接受者血清样本中的流感特异性抗体滴。第一种方法是通过血凝法确定最佳病毒抗原浓度。第二种方法通过血凝抑制来量化流感特异抗体滴。

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

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An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

35.7K 次观看.  University of Basel. 本协议描述了如何进行血凝抑制试验, 以量化流感疫苗接受者血清样本中的流感特异性抗体滴。第一种方法是通过血凝法确定最佳病毒抗原浓度。第二种方法通过血凝抑制来量化流感特异抗体滴。

视频: An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

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 ...

科研

免疫与感染

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, ...

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 ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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

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

中和流感病毒单克隆抗体逃逸变种的产生

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

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

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

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

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

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

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 ...

<meta charset="utf8"></head><body>简化 4-HAU 抗原制备，实现准确的 NDV 抗体检测

Optimization of Antigen Preparation for Hemagglutination Inhibition Assay of Newcastle Disease Virus Serology

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article introduces an optimized method for preparing 4 hemagglutination units (4-HAU) antigen solution, a key component of the hemagglutination inhibition assay (HI) used in NDV serological detection. Unlike conventional methods, which involve time-consuming and undefined back-titration and adjustment steps, this approach streamlines this process by accurately measuring the HA titer using an initial series of dilutions (1:3, 1:5, 1:7, and 1:9). We also provide a specific method for adjusting or reformulating based on the back-titration results, reducing the need for repeated back-titrations. In addition, we evaluated the effect of the accuracy of the 4-HAU antigen solution on serum HI titer and found that when the titer of the 4-HAU antigen was lower than 3, it resulted in the appearance of false-positive HI samples. By providing an accurate method and minimizing computational tasks, this approach increases test efficiency and reliability, contributing to improved disease surveillance and control in poultry populations.

<meta charset="utf8"></head><body>新城疫病毒血清学血凝抑制测定抗原制备的优化

This article presents a simplified method for preparing 4 hemagglutination units antigen for Newcastle disease virus serological testing. By accurately determining the hemagglutination titer of the antigen, combined with a more rigorous back-titration method, and a well-defined adjustment process, it enhances test efficiency, reduces false positives, and improves disease surveillance in poultry.

新城疫病毒血清学血凝抑制测定抗原制备的优化

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article ...

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Source: Leon, P. E., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56067">Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

针对中和抗体的流感病毒逃逸变异的产生

NOTE: HA-specific antibodies that inhibit viral replication can generally be categorized into i) ones that bind on or adjacent of the receptor binding ...

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

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此视频中的章节

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

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

中和流感病毒单克隆抗体逃逸变种的产生

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

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

新城疫病毒血清学血凝抑制测定抗原制备的优化

新城疫病毒血清学血凝抑制测定抗原制备的优化

新城疫病毒血清学血凝抑制测定抗原制备的优化

针对中和抗体的流感病毒逃逸变异的产生