Name: a high-throughput electrochemiluminescence 7-plex assay simultaneously screening for type 1 diabetes and multiple autoimmune diseases
Uploaded: 2020-05-29T14:00:00.000+00:00
Duration: 6 min 50 s
Description: Watch this Scientific Journal Video about A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases at JoVE.com

这项工作得到了NIH拨款DK32083，JDRF拨款2-SRA-2015-51-Q-R和2-SRA-2018-533-S-B的支持。

该研究方案得到了科罗拉多州多个机构审查委员会的批准。
1. 缓冲液制备
<ol><li>制作标记缓冲液（2x PBS，pH 7.9）。使用 400 mL 蒸馏去离子 （DD） 水，加入 100 mL 的 10x PBS。要调节溶液的pH值，请加入NaOH直至达到7.9。</li>
	<li>通过将 1 mg 生物素溶解到先前创建的 588 μL 标记缓冲液中，创建 3 mM 生物素。通过将 150 nmol的 Ru 磺基-NHS 溶解到 50 μL 标记缓冲液中，制成 3 mM Ru 磺基-NHS。</li>
	<li>通过取 500 mL 的 1x PBS 并向溶液中加入 5 g 牛血清白蛋白 （BSA） 来制作抗原缓冲液 （1% BSA）。准备0.5M乙酸溶液。使用Trizma碱制备1M Tris-HCl缓冲液，并将pH调节至9.0。</li>
	<li>对于包衣缓冲液（3%阻断剂A），取500 mL 1x PBS并加入15 g阻断剂A.通过将5000 mL 1x PBS与2.5 mL吐温20混合来制备洗涤缓冲液（0.05%吐温20，PBST）。通过加入 500 mL DD 水和 500 mL 含有表面活性剂的 4x 读取缓冲液 T 来创建读数缓冲液（含表面活性剂的 2x 读取缓冲液 T）。 注意:为了保持测定之间的一致性，重要的是生物素和Ru Sulfo-NHS溶液都是在标记程序之前创建的，而不是创建和存储以备将来使用。</li>
</ol>2. 分别用生物素和Ru磺基-NHS标记每种抗原蛋白
注意:要获得更有效的标记反应，请使用≥0.5mg / mL的抗原蛋白浓度。
<ol><li>计算每种抗原蛋白的摩尔数以及生物素和Ru磺基-NHS的摩尔数。在抗原蛋白中加入适量的生物素或Ru磺基-NHS抗原蛋白，按抗原蛋白与生物素或如磺基-NHS的摩尔比进行标记反应。<ol><li>对于分子量较小（≤10 kDa）的抗原，例如胰岛素原蛋白，请使用1:5的摩尔比（抗原:生物素和Ru磺基-NHS）。对于分子量较大（>50 kDa）的抗原，例如GAD蛋白，使用1:20的摩尔比。对于具有中等分子量（10-50 kDa）的抗原，请使用在1:5-1:20之间调整的摩尔比。</li>
	</ol></li>
	<li>对于生物素和Ru Sulfo-NHS，将每个抗原重量除以其相应的分子量，以获得每个抗原摩尔数。将摩尔数除以浓度以获得生物素的体积。对Ru Sulfo-NHS重复此操作。</li>
	<li>使用适当的摩尔比将抗原蛋白与生物素混合，在步骤2.2中测定。然后对Ru Sulfo-NHS做同样的事情。 注意:为了提高标记反应的效率，缓冲液系统中的任何还原性化学物质（如Tris或甘氨酸）都需要通过施胶离心柱交换到pH 7.9的2x PBS缓冲液中。生物素和Ru Sulfo-NHS的标记协议是相同的。</li>
	<li>用铝箔覆盖反应管，并在室温（RT）下孵育1小时。用箔纸覆盖反应管的原因是生物素和Ru磺基-NHS试剂都对光敏感。</li>
	<li>在反应管孵育时灌注2 mL或5 mL离心柱（离心柱的大小由上传到色谱柱上的体积决定）。用2x PBS缓冲液填充离心柱，然后每次以1，000 x g 离心2分钟，共三次。</li>
	<li>反应管完成孵育后停止标记反应。要停止反应，通过将标记的抗原蛋白通过离心柱一次来纯化标记的抗原蛋白。然后将色谱柱以1，000 x g 离心2分钟。</li>
	<li>通过将存在的抗原蛋白的量除以最终体积来计算总标记抗原浓度。每管等分50μL纯化的标记抗原蛋白，并将等分试样储存在-80°C以备长期使用。 注意:重要的是要注意，每次离心柱通过抗原蛋白时，都会有大约90-95%的保留率。</li>
</ol>3.确定测定（棋盘测定）中两种标记抗原的最佳浓度和比例
注意:由于该7-Plex测定中的ECL-IAA测定需要对血清样品进行酸处理，因此每种抗原的棋盘测定在与标记的抗原混合物孵育之前必须经过此步骤。
<ol><li>在运行多重测定之前，分别对每种抗原应用棋盘测定。步骤 3.2-3.6 将使用 GAD65 作为示例。</li>
	<li>计算标记的GAD65蛋白的稀释度。生物素化GAD65的第一个混合物溶液的推荐目标浓度为2000 ng / mL，Ru磺基-NHS标记的GAD65为1000 ng / mL。如果储备溶液中生物素化和Ru磺基-NHS标记的GAD65的浓度均为1.0μg/ μL，则560μL工作溶液中生物素化GAD65所需的体积将为1.12μL，700μL工作溶液中标记的GAD65的Ru磺基-NHS所需的体积将为0.7μL。</li>
	<li>将 1.12 μL 生物素化 GAD65 蛋白与 240 μL 链霉亲和素偶联接头 1 混合在一个试管中和 160 μL 1% BSA 中。将混合物在室温下孵育30分钟。向管中加入 160 μL 终止溶液，并将混合物在室温下再孵育 30 分钟。</li>
	<li>进行连续稀释。将 280 μL 混合物放入新管中，加入 280 μL 终止液以 1:2 稀释。准备几个新管。重复此步骤以运行生物素标记的GAD65抗原的水平连续稀释（参考先前的出版物12）。</li>
	<li>将 0.7 μL Ru 磺基-NHS 标记的 GAD65 （1 μg/μL） 蛋白与 700 μL 终止溶液混合。然后将 350 μL 混合物放入新试管中，加入 350 μL 终止液以 1:2 稀释。准备几个新管，重复此步骤以对Ru磺基-NHS标记的GAD65抗原进行垂直连续稀释。</li>
	<li>制备两个血清样品，一个样品对 GADA 高度阳性，一个样品对 GADA 呈阴性，每个样品的体积为 0.75 mL。将 15 μL 阳性血清等分到 96 孔 PCR 板左半部分的每个孔中。将 15 μL 阴性血清等分到 96 孔 PCR 板右半部分的每个孔中。</li>
	<li>向每个孔中加入 18 μL 0.5 M 乙酸并混合。在室温下孵育45分钟。 准备一个新的96孔PCR板。根据系列稀释液，向每个孔中加入 17.5 μL 生物素标记的抗原和 17.5 μL 的 Ru Sulfo-NHS 标记的抗原（参见之前的出版物12）。</li>
	<li>继续步骤5.2至9.1中描述的其余测定步骤。</li>
	<li>确定高正样本的信号与相应的负样本信号的比值。通过确定正负信号比例最高或接近最高比率的点，选择生物素标记抗原和Ru Sulfo-NHS标记抗原的最佳浓度。在此比率计算中，请考虑从负样本获得的低背景信号。 注意:来自棋盘测定的Ru Sulfo-NHS和生物素标记抗原蛋白的最佳浓度如下所示:GAD65为30 ng / mL和200 ng / mL，胰岛素原为120 ng / mL和120 ng / mL，IA-2为10 ng / mL和42 ng / mL，TG为80 ng / mL和80 ng / mL，TPO为8 ng / mL和16 ng / mL， ThG 为 31 ng/mL 和 31 ng/mL，IFNα 为 12 ng/mL 和 12 ng/mL。</li>
</ol>4. 创建混合接头偶联抗原溶液
<ol><li>根据棋盘测定选择每种抗原的最佳浓度。将生物素和Ru磺基-NHS标记的抗原稀释至合理的工作浓度。</li>
	<li>将不同的接头与每种独特的生物素化抗原蛋白结合。对于一个 96 孔板测定，将 4 μL 生物素化的 GAD65、TPO、tTG、ThG、胰岛素原、IFN-α 和 IA-2 蛋白与 240 μL 链霉亲和素偶联接头 1、2、3、4、8、9 和 10 混合到单独的管中（图 1）。然后每管加入 156 μL PBS/1% BSA。将混合物在室温下孵育30分钟。</li>
	<li>向每个试管中加入 160 μL 终止溶液，并在室温下再孵育 30 分钟。从每个试管中取出 400 μL 接头偶联抗原，并将所有 7 种抗原组合在一起。加入 1.2 mL 终止液，然后将 4 μL 标记的 GAD65、TPO、tTG、ThG、胰岛素原、IFN-α 和 IA-2 抗原加入混合物中。现在抗原溶液已准备好用于测定。</li>
</ol>5. 用标记的抗原孵育血清样品
注意:由于此 7-Plex 测定中的 ECL-IAA 测定需要对血清样品进行酸处理，因此每种血清都需要在与标记的抗原混合物孵育以进行此 7-Plex 测定之前进行酸处理步骤。
<ol><li>将每孔 15 μL 血清分装用于 96 孔 PCR 板。向每个孔中加入 18 μL 0.5 M 乙酸并混合。在室温下孵育45分钟。 准备一个新的 96 孔 PCR 板并将 35 μL 抗原溶液等分到每个孔中。</li>
	<li>当板仍在孵育时，向抗原板中的每个孔中加入 13 μL 1 M Tris pH 9.0 缓冲液。在每个孔的侧面添加缓冲液以限制Tris缓冲液和抗原的混合。孵育完成后，取 25 μL 酸处理的血清，并立即将其移液到抗原板上的每个孔中。搅拌溶液并用PCR密封箔覆盖板以避免光照。</li>
	<li>在室温下，将板放在摇床上，以低速设置，1小时。之后，将板储存在4°C，让板孵育18-24小时。</li>
</ol>6. 准备多重板
<ol><li>从4°C冰箱中取出一个多重板，让板进入室温。多重板处于室温状态后，向每个孔中加入 150 μL 3% 阻断剂 A。用密封箔覆盖多重板。将板在4°C冰箱中孵育过夜。 注意:检测第 1 天包括步骤 4 至 6。</li>
</ol>7. 将血清/抗原转入多重平板
<ol><li>第二天，将纸巾放在桌子上，然后从冰箱中取出孵育多重板。清空板中的所有缓冲液。为此，将板倒置并将其拍在准备好的纸巾上，直到任何孔中都没有缓冲液。</li>
	<li>通过向每个孔中加入 150 μL PBST 来清洗多重板。如步骤 7.1 中所述，丢弃缓冲区，并重复此步骤三次。将 30 μL 血清/抗原孵育到多重板的每个孔中。用箔纸覆盖板以限制其暴露在光线下。将板放在设置为低速的摇板器上，在室温下放置1小时。</li>
</ol>8.清洗板并加入读取缓冲液
<ol><li>通过将平板倒置并甩出溶液，从多重平板中取出血清/抗原孵育物。向所有孔中加入 150 μL PBST，并通过再次将板倒置并轻弹溶液从板中取出缓冲液。重复此步骤三次。第三次洗涤完成后，向每个孔中加入 150 μL 读数缓冲液。 注意:气泡会干扰酶标仪机器准确分析印版结果的能力，应不惜一切代价避免。</li>
</ol>9. 读取板并分析数据
<ol><li>在读板器上对准备好的板进行计数，以每秒计数（CPS）为单位读取所有值。</li>
	<li>使用从酶标仪机获得的抗体水平计算测定的相对指数，公式如下: 指数值 = [CPS（样本）- CPS（负标准）] / [CPS（正标准）- CPS（负标准）]。</li>
	<li>使用已确定的临界值确定哪些抗体结果为阴性或阳性。 注意:检测第 2 天包括步骤 7 至 9。</li>
</ol>

<ol>
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W., 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=Four-color+alternating-laser+excitation+single-molecule+fluorescence+spectroscopy+for+next-generation+biodetection+assays.">Four-color alternating-laser excitation single-molecule fluorescence spectroscopy for next-generation biodetection assays.</a> Clinical chemistry. 58 (4), 707-716 (2012).</li><li>Bale, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+microsphere-based+assay+for+early+detection+of+Type+I+diabetes.">A highly sensitive microsphere-based assay for early detection of Type I diabetes.</a> Technology. 02 (03), 200-205 (2014).</li><li>Ziegler, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3+Screen+ELISA+for+High-Throughput+Detection+of+Beta+Cell+Autoantibodies+in+Capillary+Blood.">3 Screen ELISA for High-Throughput Detection of Beta Cell Autoantibodies in Capillary Blood.</a> Diabetes Technology &amp; Therapeutics. 18 (11), 687-693 (2016).</li><li>Schlosser, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+Antibody+Standardization+Program:+evaluation+of+assays+for+insulin+autoantibodies.">Diabetes Antibody Standardization Program: evaluation of assays for insulin autoantibodies.</a> Diabetologia. 53 (12), 2611-2620 (2010).</li><li>Zhao, Z., 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=A+multiplex+assay+combining+insulin,+GAD,+IA-2+and+transglutaminase+autoantibodies+to+facilitate+screening+for+pre-type+1+diabetes+and+celiac+disease.">A multiplex assay combining insulin, GAD, IA-2 and transglutaminase autoantibodies to facilitate screening for pre-type 1 diabetes and celiac disease.</a> Journal of Immunological Methods. 430, 28-32 (2016).</li></ol>

作者没有什么可透露的。

在众多针对 1 型糖尿病的国家和国际临床试验中，单一 ECL 测定检测胰岛自身抗体和乳糜泻谷氨酰胺转移酶 （TGA） 自身抗体的性能已得到证实8，9，10，11。在这些试验中，当根据现有的"金"标准RBA进行评估时，该测定提高了自身抗原检测的灵敏度和特异性。在单次ECL测定和RBA14，15，16，17之间区分高亲和力和高风险胰岛自身抗体与低风险，低亲和力信号时，可以看到增强的疾病特异性。在单一ECL检测的基础上，我们提出了一种新的高通量多重ECL自身抗体检测，使我们能够同时筛查T1D以及几种适用的自身免疫性疾病。
多重ECL检测使用多重板，可将多达10种自身抗体检测组合到一个孔中。在本研究中，将7种自身抗体测定组合在一起。自身抗体包括 3 种 IAb（IAA、GADA 和 IA-2A）、2 种自身免疫性甲状腺疾病自身抗体（TPOA 和 ThGA）、乳糜泻自身抗体 （TGA） 和 APS-1 干扰素 α 自身抗体 （IFNαA）。通常，该测定机制基于先前发表的9，10，12，18的单一ECL测定，并进行了一些修改。多重ECL检测与单一ECL检测的主要区别在于，在液相中形成的每个抗体-抗原复合物都被限制在特定的接头上（图1）。生物素标记的抗原与链霉亲和素偶联物一起孵育，链霉亲和素是用于形成特异性抗原-接头复合物的接头。抗体-抗原免疫复合物在与患者血清孵育后形成，并通过多重板每个孔上的特异性接头系统捕获到特定点上。Ru Sulfo-NHS标记的抗原被抗体捕获，同时提供电化学发光信号。酶标仪可以检测来自每个孔中的点光源的多达 10 个不同信号。为了在进行长期研究时保持板之间的自身抗体测定一致，我们建议使用相同的接头。
在建立多重ECL检测之前，需要分别在多重板上优化每种自身抗体的单次ECL检测，并在常规ECL板上针对RBA和单ECL检测进行验证。为了增强棋盘格测定，对多重板上的相关自身抗体测定，使用了高阳性患者和阴性患者样本。进行棋盘测定后，计算7种自身抗体测定中每种最理想的Ru Sulfo-NHS和生物素标记抗原浓度。下面显示了这些浓度:GAD65为30 ng/mL和200 ng/mL，胰岛素原为120 ng/mL和120 ng/mL，IA-2为10 ng/mL和42 ng/mL，TG为80 ng/mL和80 ng/mL，TPO为8 ng/mL和16 ng/mL，ThG为31 ng/mL和31 ng/mL， IFNα 13 分别为 12 ng/mL 和 12 ng/mL。将所有测定组合在一起后，可能需要根据实际多重测定的结果进一步调整棋盘测定中某些抗原的浓度。
由于IAA包含在目前的多重ECL测定中，因此在将血清与抗原孵育之前，必须对血清样品进行酸处理，如先前的研究12所述。通常，当在多重检测中加入额外的自身抗体时，检测背景会受到影响，并且来自孔中一个点的极高信号可能会通过串扰阻碍附近斑点的结果。因此，对于最高阳性样品，每个自身抗体的最大CPS需要限制为20，000个计数，或更低。据我们所知，为了减少涉及的串扰量，在设计斑点图时，应将背景较低的自身抗体与计数频率较高的斑点分开。
在每次测定中内部使用高阳性和阴性对照，以计算被测未知样品的准确指数。为了准确评估和监测测定的灵敏度，使用了设置在测定上限附近的低阳性对照。这些标准阳性和阴性对照以批量和等分试样形式创建以供长期使用，并储存在-20°C或更低温度下，以确保测定之间的一致性。出于质量保证的目的，每次检测中都运行两次样品，然后重新运行每个阳性结果，并在第二天通过新的ECL检测来确认样品。如果对第一和第二验证性测定有任何分歧，则需要进行第三次测定。在进行的三种测定中，一致的两种测定的结果（例如，+，+或-,-）决定了样品的最终结果（阳性或阴性）。
在过去十年中，许多研究小组正在寻求一种利用多重方法将多种自身抗体检测组合成一个孔以筛选大量人群的高通量检测。有一些研究正在使用不同类型的技术进行多重自身抗体检测19，20，21，22，但在研究T1D时，这些检测的灵敏度和特异性与当前的"黄金"标准RBA没有可比性。所使用的这些不同类型的平台未通过国际胰岛自身抗体标准化计划（IASP）研讨会或通过在临床试验中测试大型队列进行验证。在德国最近进行的基于一般人群的筛查中，Kronus 分发的高通量联合检测 3 Screen ICATM ELISA 正被用作一线筛查的工具，以检测三种 IAb、GADA、IA-2A 和 ZnT8A，以实现儿童 T1D23 的早期诊断。3 筛选 ELISA 测定法可在 3 个分离的孔中测量 3 种自身抗体，消耗大量血清，或在混合所有 3 种检测的单个孔中测量 3 种自身抗体。如果 3 屏幕 ELISA 测定中的一个孔呈阳性，则无法区分存在三种自身抗体中的哪一个。该测定的最大缺点是无法包括IAA测量。正如IASP讲习班所证明的那样，ELISA进行的所有IAA结果都不具有可接受的敏感性和特异性24。IAA通常是第一个出现的IAb，在幼儿中患病率很高。与IAA一起进行IAb筛查对儿童是必要的，在没有IAA评估社区T1D风险的情况下进行这种筛查是不可接受的。此外，没有已发表的研究或数据显示 Kronus IAb 试剂盒检测更具 T1D 疾病特异性，能够区分高风险和低风险 IAb。在本研究中，7-Plex ECL测定使用大量新诊断的T1D13患者进行了验证。与当前标准RBA和成熟的单一ECL检测相比，7-Plex检测能够以相同的检测特异性保持100%的阳性性（表4）。目前，与标准RBA并行的4-Plex ECL测定正在应用于正在进行的大型临床试验:儿童自身免疫筛查（ASK）研究。该试验对丹佛大都会区普通人群中的儿童进行T1D和乳糜泻筛查。与ASK研究中使用的标准RBA相比，多重ECL测定显示出出色的灵敏度和更高的疾病特异性，与我们之前使用单个ECL研究的报告相同25。此外，与相应的 4 次 ECL 和 RBA 单次检测相比，我们的 4-Plex ECL 检测表明，劳动力、成本和血清量显著减少了 70%。使用多重ECL测定，我们可以用不同的数字定制每个孔，代表不同的自身抗体（最多10种），以测试特定于特定临床位置需求的不同自身免疫性疾病。
本研究显示，对于使用多重板的多重ECL测定，观察到一些局限性。与抗原一起孵育的血清的最终稀释度无法调整，以产生组合在单个孔中的每个自身抗体测定的最佳条件。观察到来自T1D患者的9个样本（9/1026）对特定自身抗体有假阴性结果。在7-Plex ECL测定中，7个假阴性是TPOA，2个是ThGA，但在单一ECL测定和RBA中都表现出高阳性结果（图2 和 3）。在所有9个样品进一步稀释后，它们在多重板上呈阳性。这个结果是由我们所说的"prozone"现象引起的。这种现象导致样品显示假阴性结果，因为高抗体滴度会影响抗原抗体晶格的形成。在设置多重检测时，对于每种联合自身抗体，建议对具有非常高滴度的样品进行预测试，以确定用于抗原孵育的血清的可选稀释度。或者，应选择具有类似优化条件的自身抗体检测以形成组合检测，从中对每种自身抗体获得最佳检测灵敏度和特异性。在本研究中，来自健康正常对照的7个样品（7/1022）在7-Plex测定中导致多种自身抗体的假阳性，但是在运行单个ECL测定和RBA（图2 和 3）后，发现这些自身抗体在两种测定中均为阴性。对于这些小样本子集，多重平板上发生这些假阳性结果的原因目前尚不清楚。对于多重ECL检测的当前应用，所有阳性样品都重复其相应的单一ECL检测以确认阳性，从而消除了多重ECL检测中的假阳性误差。

1型糖尿病（T1D）是一种严重的慢性疾病，最常见于儿童期。目前，美国约有 140 万人患有 T1D;引人注目的是，全球T1D的发病率以每年3-5%的速度稳步增长，并且在过去二十年中翻了一番，特别是在幼儿中1，2。血液循环中的胰岛自身抗体（IAbs）是目前最可靠的生物标志物。IAbs可能在临床T1D发展前数年出现3。目前，四种主要的IAb广泛用于T1D诊断和风险筛查，包括胰岛素自身抗体（IAA），谷氨酸脱羧酶-65（GADA），胰岛素瘤抗原-2（IA-2A）和锌转运蛋白-8（ZnT8A）。这四个IAb在预测T1D发展方面同样重要。T1D 的分类最近被重新定义为存在任何 4 个 IAb 中的 ≥2 个，血糖代谢正常，作为疾病 14 期。
在年轻人的糖尿病自身免疫研究（DAISY）中，发现四分之一有T1D风险的儿童可能会进展为胰岛，乳糜泻，甲状腺或类风湿自身免疫，更引人注目的是，大约40%被诊断患有T1D的患者最终会发展为额外的自身免疫性疾病5，6，7.自身抗体的鉴定对于预测和诊断这些自身免疫性疾病至关重要，应为患者提供更好的临床护理。目前还没有简单且廉价的方法来筛查这些多种自身免疫性疾病。目前使用标准放射性结合测定（RBA）和单一自身抗体测量的筛选方法对于大规模筛选来说既费力又低效。
在这里，我们将描述新开发的简单多重ECL检测，我们已经使用单个ECL检测平台8，9，10，11对其进行了验证。多重 ECL 检测将 7 种自身抗体检测结合到一个孔中，仅使用 15 μL 血清，能够同时筛查 T1D 和多种相关自身免疫性疾病，包括乳糜泻、自身免疫性甲状腺疾病和 APS-1。当时尚未开发ZnT8A ECL测定，因此未包含在多重测定中。多重ECL检测为一般人群筛查T1D和多种自身免疫性疾病提供了出色的工具。

<table><tbody><tr><td>4 &amp;deg;C refrigerator</td><td></td><td></td><td></td></tr><tr><td>&amp;ndash;80 &amp;deg;C and -20 &amp;deg;C freezers</td><td></td><td></td><td></td></tr><tr><td>96-well Plate Shaker</td><td>Wallac - Delfi</td><td></td><td></td></tr><tr><td>96-well round bottom plate</td><td>Fisher</td><td>8408220</td><td></td></tr><tr><td>Acetic acid solution</td><td>Fisher</td><td></td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>Antigen proteins</td><td></td><td></td><td></td></tr><tr><td>Human GAD65 full length protein</td><td>Diamyd</td><td></td><td></td></tr><tr><td>Human ThG full length protein</td><td>BioMart</td><td></td><td></td></tr><tr><td>Human TPO full length protein</td><td>BioMart</td><td></td><td></td></tr><tr><td>IA-2 intracellular domain protein</td><td>BioMart</td><td></td><td></td></tr><tr><td>IFN-&amp;alpha; protein</td><td>Abcam</td><td></td><td></td></tr><tr><td>Proinsulin protein</td><td>AmideBio</td><td></td><td></td></tr><tr><td>tTG protein</td><td>DiaRect</td><td></td><td></td></tr><tr><td>Biotin</td><td>Sigma</td><td></td><td></td></tr><tr><td>Bottle-Top 500 mL , Filter Units</td><td>Fisher</td><td>0974064A or B</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A-7906</td><td></td></tr><tr><td>Distilled deionized (DD) water</td><td></td><td></td><td></td></tr><tr><td>HCl</td><td>Fisher</td><td></td><td></td></tr><tr><td>Ice maker</td><td></td><td></td><td></td></tr><tr><td>Ice trays</td><td></td><td></td><td></td></tr><tr><td>MSD Sector</td><td>Perkin-Elmer</td><td></td><td></td></tr><tr><td>Multi-channel pipette</td><td></td><td></td><td></td></tr><tr><td>NaOH</td><td></td><td></td><td></td></tr><tr><td>Paper tower</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>pH meter</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Pipettes/tips</td><td></td><td></td><td></td></tr><tr><td>Ru Sulfo-NHS</td><td>MSD (R91AN)</td><td></td><td></td></tr><tr><td>Trizma Base</td><td>Fisher</td><td>BP152-5</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P-1379</td><td></td></tr><tr><td>Uplex Development Kit</td><td>MSD</td><td></td><td></td></tr><tr><td>96-well UPlex plate</td><td>MSD</td><td></td><td></td></tr><tr><td>Blocker A</td><td>MSD</td><td>R93AA</td><td></td></tr><tr><td>Linker-Streptavidin</td><td>MSD</td><td></td><td></td></tr><tr><td>Read buffer</td><td>MSD</td><td>R92TC</td><td></td></tr><tr><td>Stop Solution</td><td>MSD</td><td></td><td></td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>ZeBa Column</td><td>Pierce</td><td>89892</td><td></td></tr></tbody></table>

a high-throughput electrochemiluminescence 7-plex assay simultaneously screening for type 1 diabetes and multiple autoimmune diseases

胰岛自身抗体（IAbs）广泛用于1型糖尿病（T1D）的诊断和预测。胰岛素 （IAA）、谷氨酸脱羧酶-65 （GADA）、胰岛素瘤抗原-2 （IA-2A） 和锌转运蛋白-8 （ZnT8A） 的四种主要 IAb 在疾病预测中同样重要。目前，高达40%的T1D诊断患者继续发展为其他自身免疫性疾病。不幸的是，目前使用单一自身抗体进行测量的筛选方法对于大规模筛选研究来说既费力又低效。我们最近开发了一种简单的多重电化学发光（ECL）测定来解决这些当前问题。该测定将所有 7 种自身抗体测试组合到一个孔中。每个孔包括三个 IAb（IAA、GADA 和 IA-2A）、甲状腺过氧化物酶 （TPOA） 和甲状腺球蛋白 （ThGA） 自身抗体以检测自身免疫性甲状腺疾病、组织谷氨酰胺转移酶 （TGA） 自身抗体（用于乳糜泻）和自身免疫性多腺综合征 （APS-1） 的干扰素 α 自身抗体 （IFNαA）;所有这些都同时筛查T1D和其他相关的自身免疫性疾病。多重ECL检测基于单个ECL检测平台，而是使用多重板将多种自身抗体检测（最多10个）组合到一个孔中。与单一ECL测定的主要区别在于，在液相中形成的每个抗体-抗原复合物通过多重板上的接头系统被限制在每个孔的特定点上。在本研究中，7-Plex ECL 测定针对标准放射性结合测定 （RBA） 和单次 ECL 测定进行了验证，使用大量新诊断的 T1D 患者和年龄匹配的健康对照，从而具有出色的测定灵敏度和特异性。

Watch this Scientific Journal Video about A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases at JoVE.com

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

我们模拟了一个简单的多重ECL检测，将7种自身抗体检测组合在一起。该测定能够同时筛查 T1D 和多种其他自身免疫性疾病，包括乳糜泻、自身免疫性甲状腺疾病和自身免疫性多腺综合征 1。

同时筛查1型糖尿病和多种自身免疫性疾病的高通量电化学发光7重检测

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

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JoVE Journal

Medicine

2.4K 次观看.  University of Colorado School of Medicine. 我们模拟了一个简单的多重ECL检测，将7种自身抗体检测组合在一起。该测定能够同时筛查 T1D 和多种其他自身免疫性疾病，包括乳糜泻、自身免疫性甲状腺疾病和自身免疫性多腺综合征 1。

视频: A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

高通量顺序酶联免疫吸附试验的急性移植物抗宿主病的生物标志物的验证

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

科研

医学

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

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

人胰岛自身抗体的电化学发光测定

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

免疫与感染

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 ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

生物学

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

针对 1 型糖尿病、乳糜泻和 COVID-19 的高通量多重筛查

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and ...