这项研究得到了爱马仕 Negm 和坦尼娅莫纳汉的支持, 并通过诺丁汉消化系统疾病中心和 NIHR 诺丁汉消化系统疾病生物医学研究中心的单独资助。

1. 制备微阵列板
<ol><li>用印刷缓冲器稀释C. 梭菌抗原 [PBS-海藻糖 (50 毫米)] 在最佳的浓度 (在运行患者血清之前预定义): 毒素 a (200 µg/毫升) 和毒素 B (100 µg/毫升), pCDTb (200 µg/毫升), 纯化本机整个 SLPs 来自核糖体 001, 002 和 027 (200 µg/毫升)。 注: 毒素 b 从毒素 b 表达菌株 CCUG 20309 (90 µg/毫升) 获得。<ol>
<li>稀释阳性对照, 裂解物白念珠菌, 破伤风毒素与印刷缓冲器在100µg/毫升。最后, 稀释纯化的人类免疫球蛋白与测试过的抗体同种序列, 开始在50µg/mL 横跨10稀释在打印缓冲区, 以创建一个校准曲线。</li>
	</ol>
</li>
	<li>将打印缓冲区中的稀释的10µL 转移到384井板中。</li>
	<li>用板封和离心机盖板, 300 x 克, 5 分钟。</li>
</ol>2. 用接触机器人打印阵列
<ol><li>使用手持式燃气燃烧器加热硅销, 每 3x 2 秒, 并在清水中冲洗 3x 3 秒。</li>
	<li>将微阵列板放入机器人的加载盒中。</li>
	<li>将 aminosilane 幻灯片放在幻灯片托盘上。 注意: 幻灯片托盘可以容纳27张幻灯片: 三行九张幻灯片。幻灯片以纵向方向排列, 从底部行的左边开始, 其余的空格用空白幻灯片覆盖, 以确保幻灯片托盘上的所有孔都被覆盖。<ol>
<li>按"确定"并检查是否已打开真空, 所有幻灯片都是安全的。在开始打印之前, 将幻灯片放在潮湿的环境中至少1小时。 注: aminosilane 幻灯片是在密封袋中提供干燥剂。打开后, 任何未使用的幻灯片都应立即返回到干燥, 以便存储到到期日期为止。</li>
	</ol>
</li>
	<li>建立合适的印刷方案, 打印出梭菌抗原。启动助教应用程序套件, 并从 "我的网格运行" 目录中打开所需的打印运行参数文件。选择四份梭菌文件, 打印出所有的梭菌抗原。</li>
	<li>当双击主窗口上的微阵列图标时, 三个选项卡式窗口将显示为 "MicroArraying 参数"。 三选项卡是 "源"、"目标" 和 "选项"。"源" 选项卡显示了微阵列中使用的样本数量的详细信息。"目标" 选项卡显示了如何加载幻灯片的详细信息, 其中应在幻灯片上打印数组并编辑幻灯片版式 (16 个子数组格式)。"选项" 选项卡显示了要使用的洗涤协议和工具的详细信息。每次完成样品后清洗。清洁样品之间的硅针, 以避免不同样品之间的交叉污染。</li>
	<li>编程选项位于助教应用程序套件工具栏上的 "选项" > "运行首选项" 下。在本节中, 有9个选项卡可供使用, 当您完成一个 tab 键移动到下一个选项卡时, 单击它。单击 "确定" 按钮将带您走出 "运行首选项"。在 "运行首选项" 的 "常规" 选项卡中, 在本节中有许多可用的复选框, 其中涉及在程序启动时仪器的行为方式。检查 "开始运行前的主要浴缸" 框, 所有三个洗涤站将经历一个短的启动序列之前的运行开始。检查 "在运行开始时清洗" 框, pin 工具将在第一次源访问之前清洗。检查 "在运行结束时清洗" 框, pin 工具将在最后一次源访问后清洗。用户可以设置洗涤周期的数量。在 "运行首选项" 的 "气候" 选项卡中, 开始加湿。我们使用的设置如下: 启动速率 55%, 运行速率 55%, 最小湿度 55%, 目标湿度60%。在达到目标湿度之前, 不要开始运行, 否则斑点将是错误的大小, 库将迅速蒸发。</li>
	<li>在助教的菜单栏中, 单击 "绿色转到" 按钮。 开始运行, 并定期观察机器人在打印运行期间, 确定一切正常。 注意: 这可以在每张幻灯片上并行分析15个样本, 因为一个子数组用作负控制。子阵的设计是由印刷蛋白 (抗原) 的数量和印刷的生物复制的数量决定的。 注: 打印完每个样品后, 头部向洗涤浴池移动, 允许将针多次浸入两个沐浴液中, 每个浴缸中含有蒸馏水和0.002% 吐温 20, 然后用超纯水冲洗别针, 然后将针脚烘干。4 s 的主要洗涤站。</li>
</ol>3. 阵列的存储
<ol><li>将打印的幻灯片存放在干燥的室温下过夜。 注意: 打印的幻灯片最多可存储一周。</li>
</ol>4. 阵列探测
<ol><li>小心地将打印的幻灯片放在16多井室格式的幻灯片夹中。 注: 分庭, 深度约7.5 毫米, 提供了一个慷慨的表面体积比, 以方便混合和洗涤步骤。</li>
	<li>使用前, 请允许所有试剂对室温进行预热。除非另行指定, 否则在室温下执行以下步骤。</li>
	<li>添加100µL 过滤5% 牛血清白蛋白 (BSAT; 使用0.2 µm 注射器过滤器) 到每个块, 覆盖的幻灯片, 并孵化他们与颤抖1小时。</li>
	<li>在120µL 的磷酸盐缓冲盐水中, 每1分钟洗一次, 每袋 20 (PBST; 0.1% 补间) 用颤抖。不要让幻灯片干燥。</li>
	<li>将血清样品在冰上解冻20分钟, 用具有背景还原成分的商用抗体稀释剂稀释每个样品 (见材料表)。<ol>
<li>根据检测到的免疫球蛋白, 优化血清样品的稀释, 如表 2所示。</li>
	</ol>
</li>
	<li>将稀释后的血清样品的100µL 转移到除一条以外的所有区块, 用作阴性对照;对这个区块, 只添加100µL 的抗体稀释剂。用柔和的搅拌1小时孵化幻灯片。</li>
	<li>在120µL PBST (0.1% 补间) 中每张幻灯片 5x 3 分钟, 每井都用震动冲洗。</li>
	<li>添加100µL 的生物素化山羊抗人抗体稀释与抗体稀释剂。<ol>
<li>按照表 2所述, 根据所测试的免疫球蛋白优化二次抗体的稀释。盖上幻灯片, 用柔和的震动孵化1小时。</li>
	</ol>
</li>
	<li>在120µL 中, 每 PBST 3 分钟的每井都要用震动来清洗。</li>
	<li>添加100µL 的链亲和素 Cy5 每块稀释 1:2000 5% BSA。用箔盖在幻灯片上, 以保护他们免受光线的震动, 同时用柔和的搅动15分钟。</li>
	<li>在120µL 的 PBST 中, 每井1分钟冲洗一张幻灯片, 然后在 PBS 中进行2洗1分钟, 仅与震动。</li>
	<li>旋转幻灯片5分钟, 在 300 x g, 以烘干它们。</li>
	<li>将幻灯片放在一个深色的盒子里, 以便在室温下进行运输和储存。</li>
	<li>继续扫描阵列, 以确保探测后信号的一致性。 注意: 但是, 探测到的幻灯片可以存储在黑暗中, 并在一个星期内扫描探测。</li>
</ol>5. 扫描阵列
<ol><li>打开激光扫描仪 (见材料表), 扫描前30分钟预热激光。上载扫描仪软件并将计算机连接到扫描仪。</li>
	<li>将带有打印面的幻灯片放到扫描仪中, 直到播放指示灯变为实心绿色。</li>
	<li>在扫描幻灯片时按下 "设置" 按钮并调整以下设置: 扫描模式, 中值;承购, 仅 635;采购模式, 手册;增益, 20;功率低;幻灯片类型, 未标记幻灯片;焦点, 自动对焦。打开自动滚动, 并将条形码读数设置为像素大小10和扫描35。</li>
	<li>将结果图像保存为16位灰度多图像 TIFF 格式。按 "导入网格" 按钮, 然后选择相应的阵列列表。 注意: 数组列表描述了子阵的大小和位置以及与每个特征指示器关联的打印物质的名称。<ol>
<li>将网格与幻灯片上的点对齐。</li>
	</ol>
</li>
	<li>通过按量化过程按钮对图像进行分析, 测量每个点的荧光信号强度, 并将结果保存为称为 GenePix 结果 ("探地") 的文本格式。 注: 探地雷达文件包含了阵列抗原靶的定位和识别变量, 同时也包括了中值荧光强度和局部背景, 代表了各点的抗体结合信号值。</li>
</ol>6. 数据分析
<ol><li>用一个叫做反向相蛋白阵列 (RPPA) 分析仪的自由基因芯片分析软件 (莓36中的 R 统计语言中的一个模块) 计算背景减法后各抗原复制的中位数。</li>
	<li>利用商业科学绘图和统计软件 (见材料表), 绘制标准曲线, 并用相关的免疫球蛋白标准曲线来插入每个抗原的信号, 以计算抗体水平。</li>
</ol>

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在本议定书中, 我们已经表明, 微阵列是一个适当的平台, 以确定体液免疫反应的C. 梭菌蛋白抗原的患者血清 (图3和6) 和商业制剂丙种球蛋白 (图 5)。我们还表明, 微阵列技术表现良好, 当与常规 ELISA (图 2), 并显示优良的重现性, 与内和间化验多变性在可接受的精确度范围内 (表 1)。
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该协议描述了一种新的和自定义的蛋白质微阵列检测和半定量的细菌应变和同种特异抗体反应的C.我们成功地使用了我们的C. 梭菌特异微阵列检测作为一个有希望的新工具的成分生物分析中的具体抗体含量的患者血清1,2, 免疫球蛋白3的准备, 并识别与 CDI4中不良结局相关的抗体特异性。我们展示了如何 biobanked 血清样品和免疫球蛋白的商业制剂可以分析在微阵列, 允许高质量的重现性分析的C.
许多健康的儿童和成人有检测到的血清 IgG 和 IgA 抗体的C. 梭菌毒素 a 和 B5,6。这些被认为是发生在婴儿期短暂暴露后, 在成年后接触到C.由于这个原因, 多克隆免疫球蛋白已被使用离标签治疗复发和暴发的 CDI7,8,9。然而, 它的明确作用和行动方式仍然不清楚。几项研究表明, 体液免疫反应的C. 梭菌毒素在疾病的呈现和结果中发挥作用。具体而言, 无症状患者显示, 血清抗毒素抗体浓度增加, 与发育症状性疾病10的患者相比。据报, 有一个明显的关联, 其中中位抗毒素抗体滴度和30天全死因死亡率11。一些报告还揭示了一个协会的预防复发和抗体反应的毒素 a, B, 和一些非毒素抗原 (Cwp66, Cwp84, FliC, FliD 和表层蛋白 (SLPs))12,13,14,15. 这些观察刺激了第一个被动免疫治疗药物靶向C. 刺杆菌毒素 B (bezlotuxumab), 最近被美国食品和药物管理局和欧洲药品批准防止经常性 CDI16的机构。使用灭活毒素或重组毒素片段的免疫接种战略目前也在开发17、18、19。这些新的治疗方法无疑将刺激对大样本大小的多抗原的体液免疫应答的评估要求。
今天, 有明显的缺乏商业可用的高通量检测, 能够同时评估细菌的应变和同种特异抗体反应的C. 梭菌抗原。目前尚无未满足的需要开发这种化验, 以促进今后的研究工作和临床应用。蛋白质微阵列是一种将大量单独纯化的蛋白质作为空间组织的斑点排列在微观滑动表面上的一种方法, 使用机器人系统, 可以是接触20或非接触印刷工具21。斑点可能代表复杂的混合物, 如细胞裂解物, 抗体, 组织组织匀浆, 内源性或重组蛋白或肽, 体液或药物22,23。
蛋白质微阵列技术比标准的内部 ELISA 技术具有明显的优势, 传统的检测方法用于评估抗C.其中包括提高检测一系列多同种特异抗体的能力, 针对更广泛的蛋白质靶点, 减少抗原、样品和试剂的体积要求, 以及增强吸收更大技术复制的数量, 除了多个内部质量控制 (QC) 措施1。因此, 微阵列更灵敏、准确、重现性强, 具有更大的动态范围。这些因素使微阵列成为一种更便宜和潜在的有利替代 ELISAs 的大规模检测已知蛋白质。然而, 微阵列技术的缺点主要是由于建立了高度纯化抗原的面板, 建立了技术平台, 导致了巨大的前置成本。
蛋白质微阵列作为临床应用的诊断和基础研究工具, 在过去的两年中得到了广泛的应用。具体应用包括蛋白质表达谱、酶基质关系的研究、生物标志物筛选、寄主-微生物相互作用分析、以及分析抗体特异性23、24、 25,26,27,28。许多新的病原体蛋白/抗原微阵列已经建立, 包括疟疾 (疟原虫)29, HIV-130, 流感31, 严重急性呼吸道综合征 (SARS)32, 病毒性出血热33, 疱疹病毒34, 结核病35。
本议定书涉及建立一个易于操作的c. 梭菌反应抗原微阵列检测, 这使得对 C 的多同种和应变特异抗体反应的准确、精确和具体量化。血清中的梭菌抗原和多克隆免疫球蛋白。在这方面, 我们包括了与 ELISA 相比可接受的微阵列检测性能的具有代表性的结果, 以及测定的精确度和重现性。该方法可进一步开发, 以分析其他临床样本, 并制定一个新的标准, 研究的分子基础的 CDI。

<table border="0" cellpadding="0" cellspacing="0" style="width:1827px;" width="1826">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BioRobotics MicroGrid II arrayer</td>
			<td style="width:296px;">Digilab, Malborough, MA, USA</td>
			<td style="width:296px;">N/A</td>
			<td style="width:836px;">Contact arrayer used to automated spotting of the antigens onto the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanner InnoScan 710.</td>
			<td>Innopsys</td>
			<td>N/A</td>
			<td>A fluorescent microarray slide scanner with a red (Cy5) laser to read the reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAPIX software version 7.2.0</td>
			<td>Innopsys</td>
			<td>N/A</td>
			<td>Measure signal intensities of the spots.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon contact pin</td>
			<td>Parallel Synthesis Technologies</td>
			<td>SMT-P75</td>
			<td>Print the samples onto the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific Nalgene Desiccator</td>
			<td>Thermo Scientific</td>
			<td>41102426</td>
			<td>To store the new and printed slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384-well plate</td>
			<td>Genetix</td>
			<td>X7022UN</td>
			<td>To prepare the antigens.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate cover</td>
			<td>Sigma Aldrich, UK</td>
			<td>CLS6570-100EA</td>
			<td>To reduce evaporation of the samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminosilane slides</td>
			<td>Schott,&#160; Germany</td>
			<td>1064875</td>
			<td>The slide of choice for printing the antigens.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide holders</td>
			<td>GraceBio Labs, USA</td>
			<td>204862</td>
			<td>Divide the slides into identical 16 subarrays. These holders are re-usable, removable, leak-proof wells .&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Candida albicans lysate</td>
			<td>NIBSC</td>
			<td>PR-BA117-S</td>
			<td>Positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetanus Toxoid&#160;</td>
			<td>Athens Research and Technology</td>
			<td>04/150</td>
			<td>Positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G (IgG), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707</td>
			<td>Purified Native Human Immunoglobulin G IgG, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G1 (IgG1), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-1</td>
			<td>Purified Native Human Immunoglobulin G1 IgG1, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G2 (IgG2), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-2</td>
			<td>Purified Native Human Immunoglobulin G2 IgG2, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G3 (IgG3), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-3</td>
			<td>Purified Native Human Immunoglobulin G3 IgG3, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G4 (IgG4), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-4</td>
			<td>Purified Native Human Immunoglobulin G4 IgG4, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A (IgA), Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701</td>
			<td>Purified Native Human Immunoglobulin A (IgA), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A1 (IgA1), Human Myeloma Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701-1M</td>
			<td>Purified Native Human Immunoglobulin A1 (IgA1), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A2 (IgA2), Human Myeloma Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701-2M</td>
			<td>Purified Native Human Immunoglobulin A2 (IgA2), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin M (IgM), Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090713</td>
			<td>Purified Native Human Immunoglobulin M (IgM), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated Goat Anti-Human IgG Antibody, gamma chain specific</td>
			<td>Vector Labs</td>
			<td>BA-3080</td>
			<td>Goat anti- human IgG (&#947;-chain specific)-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG1 Hinge-BIOT</td>
			<td>Southern Biotec</td>
			<td>9052-08&#160;</td>
			<td>Goat anti- human IgG1 biotin antibody reacts specifically with human IgG1 but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG2 Fc-BIOT</td>
			<td>Southern Biotec</td>
			<td>9060-08&#160;&#160;</td>
			<td>Goat anti- human IgG2 -biotin antibody reacts specifically with human IgG 2but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG3 Hinge-BIOT</td>
			<td>Southern Biotec</td>
			<td>9210-08&#160;&#160;&#160;</td>
			<td>Goat anti- human IgG3-biotin antibody reacts specifically with human IgG3 but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG4 pFc&#39;-BIOT</td>
			<td>Southern Biotec</td>
			<td>9190-08&#160;&#160;</td>
			<td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Human IgA, alpha chain specific, made in goat - Biotinylated</td>
			<td>Vector Labs</td>
			<td>BA-3030</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgA1-BIOT</td>
			<td>Southern Biotec</td>
			<td>9130-08</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgA2-BIOT</td>
			<td>Southern Biotec</td>
			<td>9140-08&#160;&#160;</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgM-BIOT</td>
			<td>Southern Biotec</td>
			<td>9020-08&#160;</td>
			<td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 mm syringe filter</td>
			<td>Thermo scientific</td>
			<td>723-2520</td>
			<td>Filter the 5% BSA.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich, UK</td>
			<td>A7284</td>
			<td>Use 5% BSA for blocking the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody diluent</td>
			<td>Dako, UK</td>
			<td>S3022</td>
			<td>To dilute the serum and the secondary antibody.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Cy5</td>
			<td>eBioscience</td>
			<td>SA1011</td>
			<td>Detection of the immune reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087)</td>
			<td>Toxins Group, Public Health England</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified whole C. difficile toxin B (CCUG 20309 toxin B only expressing strain)</td>
			<td>Toxins Group, Public Health England</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precursor form of B fragment of binary toxin, pCDTb</td>
			<td>University of Bath&#160;</td>
			<td>NA</td>
			<td>Produced in E. Coli from wholly synthetic recombinant gene construct. Amino acid sequence based on published sequence from 027 ribotype (http:www.uniprot.org/uniprot/A8DS70)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified native whole ribotype-specific (001, 002, 027) surface layer proteins</td>
			<td>Dublin City University&#160;</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vigam (IVIg preparation 1)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Privigen (IVIg preparation 2)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Intratect (IVIg preparation 3)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection

我们提供了一种新的高通量蛋白微阵列检测方法, 用于测定人血清中的抗梭菌抗体水平, 以及单独制备多克隆静脉注射免疫球蛋白 (球蛋白)。该议定书描述了样品制备、阵列印刷、化验程序和数据分析所涉及的方法步骤。此外, 本议定书还可进一步发展, 以纳入多种临床样本, 包括血浆和细胞培养上清液。我们展示了如何使用蛋白微阵列来确定同种的组合 (IgG, IgA, IgM), 子类 (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), 和应变特异抗体的高度纯化全C. 梭菌毒素 a 和 B (toxinotype 0, 应变新品10463, ribotype 087), 毒素 b 从一个C. 芽孢杆菌毒素 b 只表达应变 (CCUG 20309), 一种前体形式的 b 片段的二元毒素, pCDTb, ribotype 特定的整个表面层蛋白 (SLPs; 001, 002, 027) 和控制蛋白 (破伤风毒素和白色念珠菌)。在实验中, 微阵列是用C................................治疗 CDI, 联合免疫机能丧失障碍, 慢性炎症性脱髓鞘 polyradiculopathy。我们在免疫球蛋白治疗前和之后的毒素中和疗效和多同种特异抗体水平上均有显著差异。此外, 基因芯片与酶联免疫吸附试验 (ELISA) 在血清样品中抗毒素 IgG 水平有显著的相关性。这些结果表明, 微阵列可能成为一个很有希望的工具来分析抗体反应的C.随着抗原板的进一步细化和生产成本的降低, 我们预计微阵列技术可以帮助优化和选择临床上最有用的免疫疗法, 以病人特有的方式感染.

图 1展示了描述协议中主要步骤的流程图。图 2显示了与检测血清中的 IgG 和 IgA 抗毒素 A 和 B 水平的基因芯片和 ELISA 之间的显著一致的长矛相关性试验。图 3显示了不同的 IgG 和 IgA 抗体类特异抗体反应的毒素 A, 毒素 B, 二进制毒素 (pCDTb) 的患者没有腹泻, CDI 患者腹泻, 和 HC。图 4显示?...

Watch this Scientific Journal Video about A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection at JoVE.com

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

本文介绍了一种简单的蛋白质芯片方法, 用于分析人体血清中高纯度梭菌抗原7丛细胞的体液免疫反应。该协议可推广用于测定多克隆静脉注射免疫球蛋白制剂中特异抗体的反应。

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

a-protein-microarray-assay-for-serological-determination-antigen

Research

JoVE Journal

Immunology and Infection

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

10.0K 次观看.  University of Nottingham. 本文介绍了一种简单的蛋白质芯片方法, 用于分析人体血清中高纯度梭菌抗原7丛细胞的体液免疫反应。该协议可推广用于测定多克隆静脉注射免疫球蛋白制剂中特异抗体的反应。

视频: A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

科研

免疫与感染

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

大肠癌细胞表面蛋白的分析，使用一种抗体的芯片和荧光多路复用

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

跨体内迟发型变态反应检测方法的抗原特异性调节

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

培养和维护<em&gt;难辨梭状芽孢杆菌</em&gt;在厌氧环境

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

头孢哌酮治疗小鼠模型的临床相关的<em&gt;艰难梭菌</em&gt;应变R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

表征形态变化的议定书<em&gt;艰难梭菌</em&gt;应对抗生素治疗

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...