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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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?...

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><tbody><tr><td>0.20 &amp;micro;m syringe filter (25mm, regenerated cellulose)</td><td>Corning, Corning, NY</td><td>431222</td><td></td></tr><tr><td>10 mL syringe (Luer-Lok tip)</td><td>BD, Franklin Lakes, NJ</td><td>302995</td><td></td></tr><tr><td>15&amp;micro; 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><td>16% paraformaldehyde</td><td>Electron Microscopy Science, Hatfield, PA</td><td>15710</td><td></td></tr><tr><td>75 cm sq. tissue culture treated flask</td><td>Corning, Corning, NY</td><td>430641</td><td></td></tr><tr><td>Agar (powder)</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A1296</td><td></td></tr><tr><td>Baby Rabbit Complement</td><td>Cedarlane Labs, Burlington, NC</td><td>CL3441</td><td></td></tr><tr><td>Bacto Yeast Extract</td><td>BD, Sparks, MD</td><td>288620</td><td></td></tr><tr><td>BBL Brain Heart Infusion (BHI)</td><td>BD, Sparks, MD</td><td>211059</td><td></td></tr><tr><td>Blood Agar (TSA with Sheep Blood) plates</td><td>Remel, Lenexa, KS</td><td>R01198</td><td></td></tr><tr><td>Cell scraper</td><td>Sarstedt, Newton, NC</td><td>83.183</td><td></td></tr><tr><td>Costar 96 well cell culture plates (plates #2 &amp;amp; 3 for dilutions)</td><td>Corning, Corning, NY</td><td>3596</td><td></td></tr><tr><td>Cover glass</td><td>Electron Microscopy Science, Hatfield, PA</td><td>72200-10</td><td></td></tr><tr><td>Difco Nutrient Broth</td><td>BD, Sparks, MD</td><td>234000</td><td></td></tr><tr><td>Dulbecco&#039;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><td>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><td>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><td>Fluorescein isothiocyanate</td><td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td><td>F143</td><td></td></tr><tr><td>HCS Cell Mask Orange Cell Stain</td><td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td><td>H32713</td><td></td></tr><tr><td>hemocytometer (Improved Neubauer)</td><td>Hausser Scientific, Horsham, PA</td><td>3900</td><td></td></tr><tr><td>India Ink solution</td><td>BD, Sparks, MD</td><td>261194</td><td></td></tr><tr><td>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><td>Nikon Eclipse TE2000-U (inverted compound microscope)</td><td>Nikon Instruments, Melville, NY</td><td>TE2000</td><td></td></tr><tr><td>PBS without Calcium or Magnesium</td><td>Lonza, Walkersville, MD</td><td>17-516F</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, 100x</td><td>Hyclone, GE Healthcare Life Sciences, South Logan, UT</td><td>SV30010</td><td></td></tr><tr><td>petri dishes (100 x 15 mm)</td><td>Falcon, Corning, Durham, NC</td><td>351029</td><td>for agar plates</td></tr><tr><td>RAW 264.7 macrophage cell line (Tib47)</td><td>ATCC, Manassas, VA</td><td>ATCC TIB-71</td><td></td></tr><tr><td>Slides</td><td>VWR, Radnor, PA</td><td>16004-422</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich, St. Louis, MO</td><td>S5761</td><td></td></tr><tr><td>Trypan Blue Solution (0.4%)</td><td>Sigma-Aldrich, St. Louis, MO</td><td>T8154</td><td></td></tr><tr><td>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...

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

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

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

科研

免疫与感染

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

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 ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

评估纳米乳剂佐剂疫苗对耐甲氧西林 金黄色葡萄球菌 （MRSA）感染的免疫反应

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

评估纳米乳化包埋的维甲酸在小鼠中的免疫原性

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

作者聚焦:用于粘膜疫苗接种的阳离子纳米乳封装视黄酸系统的开发和评估

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

阳离子纳米乳液包封的视黄酸作为佐剂，促进 OVA 特异性全身和粘膜反应

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

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

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此视频中的章节

到哺乳动物的上皮细胞在体外细菌粘附的含量

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

宿主病原学反应及疫苗效果评价

评估细菌病原体免疫反应的声吞噬杀灭试验

体外纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

评估纳米乳剂佐剂疫苗对耐甲氧西林金黄色葡萄球菌（MRSA）感染的免疫反应

阳离子纳米乳液包封的视黄酸作为佐剂，促进 OVA 特异性全身和粘膜反应

阳离子纳米乳液包封的视黄酸作为佐剂，促进 OVA 特异性全身和粘膜反应

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

摘要

探索更多视频

此视频中的章节

到哺乳动物的上皮细胞在体外细菌粘附的含量

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

宿主病原学反应及疫苗效果评价

评估细菌病原体免疫反应的声吞噬杀灭试验

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

评估纳米乳剂佐剂疫苗对耐甲氧西林 金黄色葡萄球菌 （MRSA）感染的免疫反应

阳离子纳米乳液包封的视黄酸作为佐剂，促进 OVA 特异性全身和粘膜反应

阳离子纳米乳液包封的视黄酸作为佐剂，促进 OVA 特异性全身和粘膜反应

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

体外纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

评估纳米乳剂佐剂疫苗对耐甲氧西林金黄色葡萄球菌（MRSA）感染的免疫反应