Name: a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection
Uploaded: 2018-06-15T14:00:00.000+00:00
Duration: 9 min 12 s
Description: 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

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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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+oligonucleotide+microarray+for+sub-typing+of+Influenza+A+virus.">Universal oligonucleotide microarray for sub-typing of Influenza A virus.</a> PLoS One. 6 (4), 17529 (2011).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+acute+respiratory+syndrome+diagnostics+using+a+coronavirus+protein+microarray.">Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (11), 4011-4016 (2006).</li><li>Cleton, N. 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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><tbody><tr><td>BioRobotics MicroGrid II arrayer</td><td>Digilab, Malborough, MA, USA</td><td>N/A</td><td>Contact arrayer used to automated spotting of the antigens onto the slides.</td></tr><tr><td>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><td>MAPIX software version 7.2.0</td><td>Innopsys</td><td>N/A</td><td>Measure signal intensities of the spots.</td></tr><tr><td>Silicon contact pin</td><td>Parallel Synthesis Technologies</td><td>SMT-P75</td><td>Print the samples onto the slides.</td></tr><tr><td>Thermo Scientific Nalgene Desiccator</td><td>Thermo Scientific</td><td>41102426</td><td>To store the new and printed slides.</td></tr><tr><td>384-well plate</td><td>Genetix</td><td>X7022UN</td><td>To prepare the antigens.</td></tr><tr><td>Plate cover</td><td>Sigma Aldrich, UK</td><td>CLS6570-100EA</td><td>To reduce evaporation of the samples.</td></tr><tr><td>Aminosilane slides</td><td>Schott,&amp;nbsp; Germany</td><td>1064875</td><td>The slide of choice for printing the antigens.</td></tr><tr><td>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 .&amp;nbsp;</td></tr><tr><td>Candida albicans lysate</td><td>NIBSC</td><td>PR-BA117-S</td><td>Positive control</td></tr><tr><td>Tetanus Toxoid&amp;nbsp;</td><td>Athens Research and Technology</td><td>04/150</td><td>Positive control</td></tr><tr><td>Immunoglobulin G (IgG), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707</td><td>Purified Native Human Immunoglobulin G IgG, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin G1 (IgG1), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707-1</td><td>Purified Native Human Immunoglobulin G1 IgG1, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin G2 (IgG2), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707-2</td><td>Purified Native Human Immunoglobulin G2 IgG2, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin G3 (IgG3), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707-3</td><td>Purified Native Human Immunoglobulin G3 IgG3, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin G4 (IgG4), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707-4</td><td>Purified Native Human Immunoglobulin G4 IgG4, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin A (IgA), Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090701</td><td>Purified Native Human Immunoglobulin A (IgA), Human Plasma.</td></tr><tr><td>Immunoglobulin A1 (IgA1), Human Myeloma Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090701-1M</td><td>Purified Native Human Immunoglobulin A1 (IgA1), Human Plasma.</td></tr><tr><td>Immunoglobulin A2 (IgA2), Human Myeloma Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090701-2M</td><td>Purified Native Human Immunoglobulin A2 (IgA2), Human Plasma.</td></tr><tr><td>Immunoglobulin M (IgM), Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090713</td><td>Purified Native Human Immunoglobulin M (IgM), Human Plasma.</td></tr><tr><td>Biotinylated Goat Anti-Human IgG Antibody, gamma chain specific</td><td>Vector Labs</td><td>BA-3080</td><td>Goat anti- human IgG (&amp;gamma;-chain specific)-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgG1 Hinge-BIOT</td><td>Southern Biotec</td><td>9052-08&amp;nbsp;</td><td>Goat anti- human IgG1 biotin antibody reacts specifically with human IgG1 but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgG2 Fc-BIOT</td><td>Southern Biotec</td><td>9060-08&amp;nbsp;&amp;nbsp;</td><td>Goat anti- human IgG2 -biotin antibody reacts specifically with human IgG 2but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgG3 Hinge-BIOT</td><td>Southern Biotec</td><td>9210-08&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Goat anti- human IgG3-biotin antibody reacts specifically with human IgG3 but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgG4 pFc&#039;-BIOT</td><td>Southern Biotec</td><td>9190-08&amp;nbsp;&amp;nbsp;</td><td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>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><td>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><td>Mouse Anti-Human IgA2-BIOT</td><td>Southern Biotec</td><td>9140-08&amp;nbsp;&amp;nbsp;</td><td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgM-BIOT</td><td>Southern Biotec</td><td>9020-08&amp;nbsp;</td><td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>0.2 mm syringe filter</td><td>Thermo scientific</td><td>723-2520</td><td>Filter the 5% BSA.</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma Aldrich, UK</td><td>A7284</td><td>Use 5% BSA for blocking the slides.</td></tr><tr><td>Antibody diluent</td><td>Dako, UK</td><td>S3022</td><td>To dilute the serum and the secondary antibody.</td></tr><tr><td>Streptavidin Cy5</td><td>eBioscience</td><td>SA1011</td><td>Detection of the immune reaction.</td></tr><tr><td>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><td>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><td>Precursor form of B fragment of binary toxin, pCDTb</td><td>University of Bath&amp;nbsp;</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><td>Purified native whole ribotype-specific (001, 002, 027) surface layer proteins</td><td>Dublin City University&amp;nbsp;</td><td>NA</td><td></td></tr><tr><td>Vigam (IVIg preparation 1)</td><td>Nottingham University Hospitals NHS Trust</td><td>N/A</td><td></td></tr><tr><td>Privigen (IVIg preparation 2)</td><td>Nottingham University Hospitals NHS Trust</td><td>N/A</td><td></td></tr><tr><td>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

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

科研

环境科学

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 ...

免疫与感染

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

双色抗原微阵列产生的IgG和IgM自身抗体的同时检测

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

生物工程

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

复用荧光免疫检测方法和故障排除

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

生物学

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 ...

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

使用多分析物免疫珠测定法对Covid-19进行血清转换的动态监测

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

医学

Viral Antigen Microarray Assay to Detect Antibody Isotypes in Serum Against Viral Antigen

Source: Khan, S., et al., <a href="https://app.jove.com/v/59973">Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the process for detecting antibody isotypes in multiple human serum samples using a microarray slide printed with antigenic subtypes of influenza virus strains.

病毒抗原微阵列检测试剂盒检测血清中针对病毒抗原的抗体同种型

Source: Khan, S., et al., Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes. J. Vis. Exp. (2019)
In ...