蔡美利、查博特和弗里德兰德设计了手稿中描述的程序。蔡美利和T·普特蒙-泰勒进行了实验。D. 查博特进行了数据分析。蔡先生写了手稿。
作者感谢凯尔·菲茨出色的技术援助。
这项工作得到了国防威胁减少局授予煤层气的支持。VAXBT.03.10.RD.015，计划编号921175。
意见、解释、结论和建议是作者的意见、解释、结论和建议，不一定得到美国陆军的赞同。本出版物的内容不一定反映国防部的观点或政策，也不提及贸易名称、商业产品或组织，暗示美国政府的认可。

根据《动物福利法》、《公共卫生服务政策》和其他有关动物和涉及动物的实验的联邦法规和条例，此处描述的研究是根据机构动物护理和使用委员会批准的议定书进行的。进行这项研究的设施由国际实验室动物护理评估和认证协会认可，并遵守2011年国家研究理事会《实验室动物护理和使用指南》中规定的原则。
1. 细胞系的培养和维护，RAW 264.7
注:以下程序必须使用无菌技术执行。
<ol><li>在56°C的水浴中加热胎儿牛血清（FBS），30分钟，以灭活补充。 注:当 FBS 达到 56 °C 时，30 分钟的停用时间开始。 为了监测温度，在同一水浴中用烧杯加热类似体积的水。一旦水达到56°C，开始为FBS倒计时30分钟。或者，已热灭活的 FBS 也可在商业上使用。</li>
	<li>通过将 90% DMEM 与高葡萄糖和 10% 热灭活 FBS （v/v） 相结合，为 RAW 264.7 细胞准备介质。将L-谷氨酰胺添加到6mM的最终浓度中。 注:高葡萄糖的DMEM与4mM L-谷氨酰胺配制。将L-谷氨酰胺补充到6mM可以促进细胞的持续生长。100 U/mL青霉素和100μg/mL链霉素可以添加，以防止污染。另一种常见的木氨酸细胞系，J774A.1，也可以使用，并在类似的条件下生长。</li>
	<li>在 37 °C 的水浴中解冻冷冻的细胞瓶，并在它融化后立即从浴缸中取出。在锥形管中将内容无能地添加到 5 mL 完整介质中。倒置锥形管两次，以300 x g 旋转5分钟到颗粒细胞。</li>
	<li>使用附着在真空上的无菌巴斯德移液管来去除冷冻剂的吸附介质。在5mL的新鲜介质中重新悬生细胞颗粒。</li>
	<li>将悬浮物转移到经过组织培养的烧瓶或盘子中。加入足够的介质，完全覆盖烧瓶底部，旋转以均匀分布细胞。指示以"1"开始的通道编号。</li>
	<li>在 5% CO2 和湿度存在的情况下，在 37 °C 下孵化细胞，直到达到 95% 到 100% 的汇合度。从孵化的第二天开始，每天在显微镜下检查细胞。不要让细胞达到 +100% 的汇合度，因为这会导致细胞升空和死亡。 注:达到汇合所需的时间取决于镀多少活细胞和烧瓶的生长区域。因此，每天检查细胞是谨慎的。</li>
	<li>通过刮和稀释新鲜介质中的细胞存量，亚培养细胞允许刮擦之间至少生长两天。每个亚文化增加1个通道数。 注:通过和立即刮第二天将导致随着时间的推移更快地失去一般细胞依从性。RAW 264.7细胞只能使用到大约15通道。</li>
	<li>要板 RAW 264.7 细胞进行 OAA 检测，请更换新鲜介质，然后使用细胞刮刀轻轻刮掉烧瓶上的细胞。上上下下的移液器可以分解细胞团，以便于细胞计数。 注:将RAW 264.7亚文化的传统方法是刮擦。根据我们的经验，尝试蛋白的使用会导致 RAW 264.7 细胞随着时间的推移比刮擦更快地失去依从性。</li>
	<li>要计数细胞，稀释等量的细胞和尝试泛蓝色溶液。将 10 μL 加载到测压仪上。使用带 10 倍目标透镜的倒置复合显微镜观察和计数排除染料的活细胞数量。</li>
	<li>板 1.4 x 104可行的细胞每口井在 96 井 0.15μm 厚玻璃平底板 （板#1）.允许细胞生长3天。在执行OAA前一天更换中等。 注:经过3天的孵化，细胞数量应约为5×104/井。</li>
</ol>2. 细菌培养和制剂
注:本协议中使用的细菌物种是一种封装的毒株 ，B.无烟艾 姆斯。与该物种的所有操作都需要适当的生物安全和安全许可，并且必须在位于 BSL-3 实验室的 II 类或 III 类生物安全柜中执行。在处理细菌时，遵循BSL-3工作和使用个人防护设备的机构操作程序。
<ol><li>准备脑心脏输液 （BHI） 汤，在 1 L 水中溶解 37 g BHI 粉末，并在 121 °C 下自动拍打 15 分钟。在环境温度下存放肉汤。</li>
	<li>在水中准备 8% 的碳酸氢钠溶液，并使用 0.20μm 过滤注射器进行消毒。在4°C存储解决方案，并在1天内使用。</li>
	<li>混合 8 g 营养汤， 3 克酵母提取物和 15 克琼脂在 1 L 水准备 Nby 琼脂板。高压在121°C下15分钟。倒入培养皿，并允许凝固。4°C的存储板。 注:NBY琼脂板可以用或不含碳酸氢钠制成。在肉汤和琼脂板中加入碳酸氢钠，可诱使由封装的巴西利组成的粘膜菌落生长。液体和固体介质中碳酸氢钠的最终浓度为0.8%。</li>
	<li>通过混合 50% 高压 BHI 汤、40% FBS 和 10% 碳酸氢钠溶液 （v/v） 为 B. 无 烟素准备培养汤。 注:这种肉汤的配方是为了生产短链封装的巴西利。</li>
	<li>准备一盘 B. 无烟的 主盘， 在汤中栽培的前一天， 在 Nby agar 盘子上与孢子存量隔离。在37°C下孵化。</li>
	<li>在通风的250 mL烧瓶中接种30 mL的文化汤，并带有几个 B.无烟菌群。 注:肉汤的特定体积与表面比率（如此处所述）需要产生最大限度封装的巴西利。</li>
	<li>以 125 rpm、37 °C 和 20% CO2 和 18-24 h 的湿度摇动肉汤培养。 注:在超过37°C的温度下生长 无烟菌 可能会抑制胶囊的产生。</li>
	<li>使用印度墨水的负染色，以确保足够的封装和巴西利在固定前处于短链（1-5 巴西利/链）（见第 3 节）。</li>
	<li>要确定菌落形成单位 （CFU），在磷酸盐缓冲盐 （PBS） 溶液中连续稀释 100 μL 的培养物 1:10，并在羊血琼脂板上稀释板培养物。在37°C下孵育12-18小时。 数一数CFO，并确定每mL培养的细菌数量。 注:一旦细菌被修复，也可以在显微镜下使用测高仪进行计数。但是，不建议这样做，因为在低放大倍率下很难看到巴西利。</li>
	<li>将16%的准甲醛（PF）添加到细菌培养的整个体积中，最终浓度为4%PF，以修复杆菌。在环境温度下缓慢地在摇床上搅拌 7 天。 注:4%PF的7天孵化是基于美国医学机构标准操作程序，用于修复植物性巴西里。</li>
	<li>为了去除固定和测试不育，三次洗4 mL（10%体积）的固定细菌悬架在50mL圆锥管离心在≥3000 x g 45分钟，并使用30 mL/洗与PBS。将剩余样本存储在 4% PF 中，在 4 °C 下。 注:颗粒后，由于胶囊的存在，细菌颗粒蓬松。在洗涤过程中注意不要打扰颗粒。有必要删除所有固定，以免干扰可行性测试。如有必要，增加洗涤次数。</li>
	<li>用于生存能力测试，为 40 mL 的细菌生长介质（例如 BHI 肉汤）接种 4 mL 的洗涤悬架（≤汤体积的 10%），并在 37 °C 下孵育 7 天。在 7 天前后检查 600 nm 的光学密度，以测量浊度。</li>
	<li>在肉汤养殖中孵育 7 天后，将 100 ≥ μL 的肉汤板放到琼脂板上，再孵育 7 天。如果浊度没有增加，板块上也没有生长，原始培养物被认为是无菌的，可以转移到BSL-2实验室。 注:联邦选择剂计划22规定，灭活的无烟炭毒只有在证明样品完全不育后才能从BSL-3实验室转移。灭活的无烟杆菌的活性测试包括在肉汤中培养10%的样本7天，然后用固体介质再培养7天22，23。遵循机构准则，一旦不育，将获得转让批准。</li>
</ol>3. 负染色和光显微镜观察封装和巴西利链
<ol><li>在显微镜滑梯上将 5 μL 细菌悬浮液与 2 μL 印度墨水混合。在悬架顶部放置一个 1 或 1.5 μm 厚的盖玻璃，而不会产生气泡。 注意:指甲油可用于将盖玻璃密封在滑梯上。</li>
	<li>在光显微镜上使用 40 倍至 100 倍的油目标透镜观察细菌悬架。通过观察每个细菌周围的间隙区域（胶囊不包括印度墨水），确保囊状物被封装，并确保巴西利链短。</li>
</ol>4. 细菌的 FITC 标签
<ol><li>溶解荧光素异硫氰酸酯（FITC）在二甲基硫化物浓度为2毫克/mL。</li>
	<li>测量灭活细菌库存的体积。</li>
	<li>在PBS中清洗股票3倍，以消除固定性。</li>
	<li>在最终浓度为 75 μg/mL 时添加 FITC 解决方案。在 4 °C 下缓慢搅拌混合物 18-24 小时。</li>
	<li>以≥3000 x g、45分钟和30mL PBS/洗涤来清除多余的FITC，从而洗涤细菌。确保毛绒颗粒在清洗过程中不会受到干扰。以相同的启动音量重新暂停 Pbs 中的巴西利。</li>
	<li>在-20°C的微管中，在使用之前，在微管中存储巴西里。不要多次冻结/解冻巴西利，因为巴西利可能会失去胶囊。</li>
</ol>5. 细菌蛋白化
<ol><li>热在 56 °C 下在热块中灭活少量测试塞拉（来自 NHP），温度为 30 分钟。</li>
	<li>用PBS 2x解冻和清洗FITC标签的细菌，以15000 x g 的颗粒3-5分钟。以相同的起始量在PBS中重新暂停。</li>
	<li>在96井圆形底板（板#2），在细胞介质中连续稀释测试塞拉。在另一个 96 井圆形底板（板#3）中，在每个油井中加入 10 μL 的稀释测试塞拉。 注:在每一个盘子中，PBS和普通猴子塞拉被纳入稀释测试塞拉作为阴性对照。我们还选择了一种测试血清作为正对照，以生成一个标准曲线，使不同天进行的所有检测正常化。阳性对照测试血清被任意选择，因为它是丰富的。</li>
	<li>要板#3，添加 10 μL 新鲜重组的小兔子补充。 注:一旦重组，丢弃剩余的兔宝宝补充:不要重新冻结和解冻。</li>
	<li>要板#3，添加适当的数量 FITC 标记的巴西里，感染的倍数为 1:20。例如，添加包含 5 x 104 x 20 bacilli 的体积，用于 5 x 104 个单元。板#3中每口井的顶端，细胞介质的相应体积为 105 μL。 注:含有细胞的板#1接收100μL的蛋白化细菌，其余5 μL为空体积。我们以重复测试 Sera 测试的每一个稀释。</li>
	<li>孵化板在37°C下#3 30分钟，存在5%的二氧化碳 ，湿度为巴西利。</li>
</ol>6. 哺乳动物细胞的粘性分析和荧光标签
<ol><li>使用前将所有试剂保持在37°C。</li>
	<li>将含有附体细胞的板#1放置在 37 °C 热块上。</li>
	<li>使用多通道移液器从井中取出废料，并快速从板#3中替换 100μL 的蛋白化细菌。确保在管道过程中井中不会产生气泡。孵化板#1在37°C与5%的二 氧化碳和湿度为30分钟的坚持。</li>
	<li>用 150μL PBS 在 37 °C 热块上清洗每口井 5 倍，以去除未连接的巴西利。 注意:必须小心，确保细胞在洗涤过程中不会意外分散。</li>
	<li>稀释 16% PF 与 PBS 1:4 使 4% PF. 修复细胞与 4% PF 1 小时通过添加 150μL 到每个井。用PBS清洗电池2倍。</li>
	<li>在 10 mL PBS 中混合 2 μL HCS（高含量筛选）橙色细胞污渍。将150μL的这种染色溶液添加到固定细胞中，并在环境温度下孵育30分钟。</li>
	<li>用PBS洗2次井。</li>
	<li>存放在4°C，直到显微镜检查。</li>
</ol>7. 显微镜
注:一般来说，高通量自动荧光显微镜将促进OAA的成像。如果没有自动系统，可以手动拍摄粘附巴西利的荧光图像。在这项研究中，20，坚持巴西利和细胞单层图像使用蔡司700激光扫描显微镜系统与专用设备:我们的系统由蔡司 Axio 观察者 Z1 倒置显微镜组成，配备自动舞台、定焦模块、40 x NA 0.6 目标、Axio Cam Hrc 相机和 Zen 2012 （蓝色版） 软件。获得的图像是广域图像，而不是合金图像。以下协议旨在作为适用于各种自动显微镜系统的基本显微镜设置。
<ol><li>在环境温度下孵化板#1 30分钟。打开显微镜和相关设备。 注:环境温度的适应可防止在成像过程中在玻璃板上形成凝结。此外，它防止因温度变化而去除。</li>
	<li>将板放在舞台上，使用明亮的字段或相位对比聚焦于细胞。</li>
	<li>选择96井格式作为板设置。选择通道设置。 注:FITC的峰值激发和发射波长为495/519 nm:HCS橙色细胞污渍为556和572 nm。其他荧光片可能根据显微镜上的滤镜使用。</li>
	<li>选择"磁贴模式"的设置。指示要获取的图像数量。 注:平铺功能允许在样本井中捕获多个位置，并可以设置为以平铺或随机格式获取图像。我们映像了每口井10个随机区域。</li>
	<li>将获取模式更改为"黑白"或"单色"，并将≥2x2装箱。 注:将垃圾箱从 1x1 设置为 2x2 或 4x4 可提高显微镜速度，但也会降低图像的分辨率。对于 OAA 来说，当检测同时列举巨噬细胞和附体细菌时，使用这些设置就足够了。</li>
	<li>打开自动对焦或对焦模块。指示执行自动对焦功能的频率。如果可用，则打开 Z 堆栈功能。指示将捕获细胞单层和附体 bacilli 厚度的 Z 堆栈数量。 注:选择的 Z 堆栈数量将增加显微镜时间，但将确保每个 Z 堆栈拍摄具有正确对焦的图像。</li>
	<li>指定文件夹以将图像保存到。运行自动成像程序。</li>
</ol>8. 数据收集和分析
<ol><li>按图像计算附体巴西利和哺乳动物细胞的数量。将每链巴西里数为一种细菌。</li>
	<li>绘制阳性对照测试血清和滴定（例如，1:10 稀释为 10 滴定单位）的巴西利/细胞，以在适当的统计程序中生成标准曲线。</li>
	<li>使用非线性回归曲线拟合分析正对照测试血清。然后使用阳性对照测试血清的标准曲线分析剩余样品，以确定相应的滴定器。 注:在确定滴度时，通常最准确地选择标准曲线中间附近的样品稀释。</li>
	<li>计算每个疫苗组样本的几何平均值。通过最不显著的差异 ANOVA 分析计算日志转换滴度的 P 值。</li>
</ol>

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作者没有可能构成利益冲突的商业或其他关联。

胶囊疫苗已被证明对许多细菌病原体有效，许多疫苗获准用于人类25、26、27。这些疫苗通过产生针对胶囊的抗体而起作用，其中许多研究使用OPKA来显示抗体13、14、16、28、29的蛋白细胞功能。出于对生物安全和工作的便利性，我们开发了一个OAA来评估接种无烟胶囊结合体的动物的抗体。无烟杆菌上的胶囊的存在可以防止粘附和噬细胞增多症30，但从接种疫苗的动物中分离出的血清会导致附着物附着在巨噬细胞20，21。
对于OAA来说，坚持活动，而不是内化和杀戮，被用来量化行动活动。这为OAA提供了几个优势。我们能够利用被杀死的甲醛，荧光标记封装的巴西里，而不是活的生物体。醛固定和荧光标签对细菌表面的改变并没有改变细菌对巨噬细胞的整体依从性:固定和标记的巴奇利并不比活巴西利更坚持 （数据未显示） 。封装程度可能因文化而异，尽管生长条件相似，但会影响整体依从性。因此，使用单个灭活细菌库允许在不同日期和不同实验组进行实验的标准化。这对于比较本作品中使用的 20 个 NHP 的塞拉和将在我们即将进行的研究中生成的塞拉是有价值的。最后，大多数巨噬细胞，包括RAW 264.7细胞，对活病原体31产生的炭热致命毒素敏感，使得使用活杆菌本身就有问题。
RAW 264.7细胞的使用也促进了标准化。我们最初在 OPKA、HL-60 细胞中使用了常用的细胞系。然而，我们发现很难区分他们与粒细胞与二甲基酰胺，如其他人报告28，32。RAW 264.7 细胞具有化学分化和粘附性，因此更适应基于显微镜的 OAA 的优势。这种细胞系已被用于许多噬细胞病研究与细胞内病原体33，34，以及B.无烟31。RAW 264.7细胞的使用，这是从小鼠，也是有利的，因为小鼠Fc受体是混杂在其结合特异性IgG从其他物种35。这使我们能够用穆林细胞线评估来自NHPs的抗胶囊抗体。
炭热虽然罕见，但在世界和美国的一些地区很普遍。一个担忧是，从美国采购的FBS可能已经含有抗胶囊抗体，因为动物在土壤中暴露于 无烟孢 或其他PGGA产生的微生物。为了解决这个问题，我们使用的 FBS 批次进行了预先测试。我们发现，与 DMEM 相比，添加热灭活 FBS 确实增加了依从性（未显示数据）。然而，它并没有增加坚持相比，热灭活正常塞拉从豚鼠，猴子，老鼠或人类（数据未显示）。因此，我们使用的 FBS 批次由于暴露而不包含抗胶囊抗体。此外，它也不会含有抗胶囊抗体，因为动物接种了未封装的 无烟炭热 斯特恩，这种菌株缺乏胶囊生产所需的pXO2质粒。测试的 FBS 批次用于所有后续 OAA。
该技术的一个局限性是实验室人员对巴西利和细胞进行计数的任务，这需要大量的时间和精力。使用具有这种分析类型的软件可以纠正这种情况:这个软件当时没有提供给我们。然而，由于人工计数是必要的，一个关键步骤是去除或洗掉无关和未连接的巴西里，以促进任务。还需要测试导致低背景噪音的试剂。OAA需要一个补充的来源，因为它提高了操作性36。因此，我们测试了各种补充来源，包括豚鼠，人和小兔补充。我们选择小兔子补充我们的检测，因为我们发现，它没有弱，多特异性活动对异性恋物抗原，它通常用于OPKA研究14，15，37，它是现成的商业。
我们发现OAA是定量的和高度可重复的。我们用它来补充涉及连体结合检测（如 ELISA）的研究，这些研究仅量化 IgG 总水平，但不区分功能性和非功能性抗体。OAA可用于补充其他功能检测（例如血清杀菌剂活性和毒素中和检测），以显示疫苗产生的抗体16，38的不同功能。OAA的发展增加了我们的免疫原性研究，以评估疫苗和治疗方法。

病原体的识别、坚持、内化和退化是噬菌体细胞增多症1的组成部分，噬菌体是伊利亚·梅奇尼科夫在1883年第2、3年首次描述的宿主先天免疫反应的显著途径。噬细胞白细胞以及免疫系统的其他细胞在选择靶点时具有高度歧视性:他们能够区分"传染性非自我"和"非传染性自我"通过病原体相关的分子模式，通过他们的模式识别受体（PRR）4，5的剧目。宿主识别病原体也可能发生与宿主蛋白的结合，如补充和抗体6。这个过程，称为opsoning，用这些分子涂覆病原体，在与噬菌体细胞6的opsonic受体（例如补充和Fc受体）结合后增强内化。病原体要粘附在噬细胞上，就必须将多种受体与其同质配体集体结合。只有这样，坚持才能触发和维持主机单元内的信号级联，以启动内化6。
由于噬细胞病在清除病原体和预防感染方面的重要性，细胞外病原体已经开发出许多方法来颠覆这一过程，以延长其生存时间。一种重要的策略是生产一种离子聚合物（例如多糖或多氨基酸）胶囊，这种胶囊由于其电荷而具有抗噬菌性，免疫力差，并且保护细菌包络上的分子免受PRR6、7的侵害。病原体，如隐球菌新福尔曼和肺炎链球菌有由糖化物组成的胶囊，而葡萄球菌表皮和一些杆菌物种产生多ɣ谷氨酸（PGGA）7，8。然而，其他病原体产生的胶囊类似于非传染性自我。例如，链球菌和B.cereus的致病菌株有一个透明质酸胶囊，它不仅是抗噬菌体，而且可能不被免疫系统9，10识别为异物。
胶囊与载体蛋白的结合，将他们从贫乏的T独立抗原转化为高致免疫性T依赖抗原，可诱导高血清抗胶囊抗体滴度11，12。该策略用于针对肺炎、流感嗜血杆菌和奈塞里亚脑膜肽11的许可疫苗。抗胶囊抗体的手术活动通常由奥普索诺-法果细胞杀伤性检测（OPKA）13、14、15、16进行评估。这些检测测试功能抗体是否可以触发噬细胞增多症和杀死14。然而，使用带有传染性病原体的OPKA，如一级生物选择剂和毒素（BSAT），包括B.无烟体17，是危险的，并带来安全风险:这些检测需要广泛处理选择的代理。选择剂处理只能在受限生物安全级别 3 （BSL-3） 实验室进行:由于必须遵循许多安全和安保预防措施，这些领域的工作需要长期的操作程序。BSL-3实验室通常也没有配备用于OPKA工作的专用设备，如显微镜和细胞计。因此，我们开发了一种基于使用灭活细菌18，19的替代检测方法。我们称之为一种不依赖于内化和杀戮的opsono-依从性检测（OAA），作为检测输出:相反，对非活化灭活病原体的坚持被用作噬细胞病的指数。机械上，OAA是一个合适的替代品，因为坚持发生先验，并与内化和细胞内杀密切交织在一起。从生物安全的角度来看，OAA 是首选，因为它需要最少的传染性剂处理，实验时间比 OPKA 短，并且可以在 BSL-2 实验室中进行，在产生和转移灭活病原体库存后进行。
我们证明利用OAA来检查在非人类灵长类动物（NHPs）的血清中发现的抗胶囊抗体的opsonic功能，这种抗胶囊抗体是用胶囊结合而接种的[即从B.无烟菌结合到奈西里亚脑膜肽的外膜蛋白复合物（OMPC）的PGGA]20。血清通配的巴西利被孵育与附体鼠标巨噬细胞线，RAW 264.7。固定后，细胞单层和附体巴西利通过荧光显微镜成像。与对照血清20相比，当用用胶囊结合接种的NHPs的血清孵化时，细菌依从性增加。坚持与炭热挑战20，21的生存有关。因此，OAA的使用具有抗胶囊抗体的功能，大大方便了疫苗候选剂的测试。

<table border="0" cellpadding="0" cellspacing="0" style="width:1520px;" width="1520">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">0.20 &#181;m syringe filter (25mm, regenerated cellulose)</td>
			<td style="width:483px;">Corning, Corning, NY</td>
			<td style="width:267px;">431222</td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mL syringe (Luer-Lok tip)</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15&#181; 96 well black plates (plate #1 for imaging)</td>
			<td>In Vitro Scientific, Sunnyvale, CA</td>
			<td>P96-1-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% paraformaldehyde</td>
			<td>Electron Microscopy Science, Hatfield, PA</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">75 cm sq. tissue culture treated flask</td>
			<td>Corning, Corning, NY</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar (powder)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Baby Rabbit Complement</td>
			<td>Cedarlane Labs, Burlington, NC</td>
			<td>CL3441</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Yeast Extract</td>
			<td>BD, Sparks, MD</td>
			<td>288620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BBL Brain Heart Infusion (BHI)</td>
			<td>BD, Sparks, MD</td>
			<td>211059</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blood Agar (TSA with Sheep Blood) plates</td>
			<td>Remel, Lenexa, KS</td>
			<td>R01198</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell scraper</td>
			<td>Sarstedt, Newton, NC</td>
			<td>83.183</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Costar 96 well cell culture plates (plates #2 &#38; 3 for dilutions)</td>
			<td>Corning, Corning, NY</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover glass</td>
			<td>Electron Microscopy Science, Hatfield, PA</td>
			<td>72200-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Difco Nutrient Broth</td>
			<td>BD, Sparks, MD</td>
			<td>234000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose</td>
			<td>Gibco, Thermo Fisher Scientific, Waltham, MA</td>
			<td>11965-092</td>
			<td>contains 4500 mg/L glucose, 4 mM L-glutamine, Phenol Red</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EVOS FL Auto Cell Imaging System (fluorescence microscope)</td>
			<td>Life Technologies, Thermo Fisher Scientific, Waltham, MA</td>
			<td>AMAFD1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Hyclone, GE Healthcare Life Sciences, South Logan, UT</td>
			<td>SH30071.03</td>
			<td>not gamma irradiated, not heat inactivated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein isothiocyanate</td>
			<td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td>
			<td>F143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCS Cell Mask Orange Cell Stain</td>
			<td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td>
			<td>H32713</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hemocytometer (Improved Neubauer)</td>
			<td>Hausser Scientific, Horsham, PA</td>
			<td>3900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">India Ink solution</td>
			<td>BD, Sparks, MD</td>
			<td>261194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L- glutamine (200 mM)</td>
			<td>Gibco, Thermo Fisher Scientific, Waltham MA</td>
			<td>25030081</td>
			<td>supplement medium with additional 2mM L-glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Eclipse TE2000-U (inverted compound microscope)</td>
			<td>Nikon Instruments, Melville, NY</td>
			<td>TE2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS without Calcium or Magnesium</td>
			<td>Lonza, Walkersville, MD</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin Solution, 100x</td>
			<td>Hyclone, GE Healthcare Life Sciences, South Logan, UT</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petri dishes (100 x 15 mm)</td>
			<td>Falcon, Corning, Durham, NC</td>
			<td>351029</td>
			<td>for agar plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RAW 264.7 macrophage cell line (Tib47)</td>
			<td>ATCC, Manassas, VA</td>
			<td>ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slides</td>
			<td>VWR, Radnor, PA</td>
			<td>16004-422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue Solution (0.4%)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss 700 Laser Scanning Microscopy (confocal microscope)</td>
			<td>Carl Zeiss Microimaging, Thornwood, NY</td>
			<td>4109001865956000</td>
			<td></td>
		</tr>
	</tbody>
</table>

opsono-adherence assay to evaluate functional antibodies in vaccine development against bacillus anthracis and other encapsulated pathogens

蛋白石粘性检测是一种功能性检测，它列举了细菌病原体与专业噬细胞的附着。因为坚持是法戈西病和杀戮的必要条件，因此测定是奥普索诺-法细胞杀菌检测的替代方法。opsono-依从性检测的一个优点是可以选择使用灭活病原体和哺乳动物细胞系，从而允许跨多个实验进行标准化。在检测中使用灭活病原体也有助于生物安全3级传染性剂和其他致命病原体的工作。在我们的工作中，opsono-依从性检测用于评估抗体的功能能力，从用炭热胶囊疫苗免疫的动物血清，到诱导固定 杆菌无烟杆菌 与小鼠大噬细胞系RAW 264.7的粘合。自动荧光显微镜用于捕捉巴西利粘合巨噬细胞的图像。增加依从性与血清中是否存在抗胶囊抗体有关。表现出高血清抗胶囊抗体浓度的非人类灵长类动物受到保护，免受炭热挑战。因此，蛋白石粘性检测可用于阐明血清中抗原特异性抗体的生物功能，评估疫苗候选物和其他治疗方法的疗效，并作为免疫力的可能相关性。

本节显示了在OAA实验中收集的具有代表性的显微图，以及显示OAA可用于检查抗体的生物功能的结果。在这里，该检测被成功地用于评估炭热疫苗候选的疗效。验证囊体上的封装状态至关重要，因为很少或根本没有封装会导致它们粘附在宿主细胞上，从而产生高背景。 图 1 是 B. 无烟艾 姆斯的图像，上面印有印度墨水，以检查封装。OAA 要求照亮多个相邻的视场，如果使用共光显微镜，则需要多个堆栈或部分。因此，有必要验证，巴西利既不是太暗，也不是照片漂白太快。 图 2 是标有 FITC 的巴西利图像。 图 3 显示附着在细胞单层上的 B. 无烟艾 姆斯的最大投影图像。这些是为OAA计算和得分的具有代表性的观点领域。与PGGA-OMPC抗体一起孵育的巴西利附着物的增加是由于存在抗胶囊抗体，使巴西利具有灵丹妙药。 图 4 显示，仅接种 10 和 50μg PGGA-OMPC 的 NHP 血清滴定器就明显高于 OMPC 和 PGGA 的滴答声。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60873/60873fig1.jpg"> 图 1.固定 B. 无烟艾 姆斯的印度墨水图像。 请注意，由于胶囊的存在，巴西利周围的间隙区域。酒吧=10微米。该图像是在 EVOS FL 自动显微镜系统上拍摄的，该系统具有 100 x 1.4 数光圈 （NA） 油目标透镜。 <a href="https://www.jove.com/files/ftp_upload/60873/60873fig1large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60873/60873fig2.jpg"> 图2。FITC 标记固定 B. 无烟艾姆斯的 图像。 （左）差异干扰对比度图像:荧光图像假彩色绿色（中间）:合并（右）。酒吧=10微米。在蔡司 700 LSM 康菲科显微镜上拍摄了 40 x 1.3 NA 油目标透镜的康福克图像。 <a href="https://www.jove.com/files/ftp_upload/60873/60873fig2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60873/60873fig3.jpg"> 图3。 B. 无烟艾 姆斯的荧光图像粘附在 RAW 264.7 细胞单层上。 巴西利通过接种的 Nhps 的测试血清进行手术， 并仅通过（A）10μg PGGA - OMPC （B）50μg PGGA- OMPC 、（C）50 μg PGGA 或（D）OMPC 进行提升。绿色 =B.无烟艾 姆斯，红色=RAW 264.7细胞。酒吧=20μm。在蔡司阿希奥观察者Z1显微镜与40×0.6客观镜头和Axio凸轮HRC相机上拍摄了广域图像。 <a href="https://www.jove.com/files/ftp_upload/60873/60873fig3large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60873/60873fig4.jpg"> 图4。NHP 的奥索诺坚持滴度接种了 10 μg PGGA-OMPC、50 μg PGGA-OMPC、仅 50 μg PGGA 或仅 OMPC。绘制了每组 5 种动物的几何方法。错误条表示SEM.p≤0.001仅与PGGA相比。p值是使用 ANOVA 与 LSD 弹射后测试计算的。这些数据最初发表在查博特等人，2016年20日。<a href="https://www.jove.com/files/ftp_upload/60873/60873fig4large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens at JoVE.com

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

opsono-依从性测定是分析抗体在疫苗开发中的opsonic功能的替代方法。

Opsono-坚持性检测，以评估疫苗开发中针对 无烟杆菌 和其他封装病原体的功能抗体

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...

opsono-adherence-assay-to-evaluate-functional-antibodies-vaccine

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

3.3K 次观看.  United States Army Medical Research Institute of Infectious Diseases. opsono-依从性测定是分析抗体在疫苗开发中的opsonic功能的替代方法。

视频: Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

科研

免疫与感染

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

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 ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

利用人类诱导的多能干细胞衍生的肠道有机体研究和改性上皮细胞对沙门氏菌和其他病原体的保护

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

使用流感抗原微阵列测量病毒亚型血清抗体的广度

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

使用基于路西法抹去的生长测定，确定化学抑制剂对细胞内弓形体贡迪生长的抑制效率

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Opsono-坚持性检测，以评估疫苗开发中针对 无烟杆菌 和其他封装病原体的功能抗体

Opsono-坚持性检测，以评估疫苗开发中针对无烟杆菌和其他封装病原体的功能抗体