这项工作得到了 NIH 赠款 DK32083, JDRF 赠款 2-2015-51 Q R 的支持。

1. 缓冲准备
<ol><li>标签缓冲 (2x pbs, pH 值 7.9): 在400毫升的蒸馏去离子 (DD) 水, 添加100毫升的 10x PBS, 调整 pH 值为7.9 与氢氧化钠。</li>
	<li>3毫米生物素: 将1毫克的生物素溶于588µL 的标记缓冲液中。</li>
	<li>3毫米的磺标签: 在50µL 标签缓冲器中溶解 150 nmol 的磺标记。</li>
	<li>抗原缓冲 (5% BSA): 500 毫升 1x PBS, 添加25克牛血清白蛋白 (bsa)。</li>
	<li>准备0.5 米醋酸溶液。</li>
	<li>1.0 米的三盐酸缓冲液 pH 值 9.0: 准备1米三盐酸缓冲液, 并将 ph 值调整为9.0 与 hcl。</li>
	<li>涂层缓冲器 (3% 阻挡剂 a): 在500毫升 1x PBS 中, 添加 15 g 的阻滞剂 a。</li>
	<li>洗涤缓冲器 (0.05% 吐温 20, PBST): 在5000毫升 1x PBS, 增加2.5 毫升的补间20。</li>
	<li>读取缓冲区 (2x 读取缓冲 t 与表面活性剂): 在 DD 水的500毫升, 添加500毫升4x 读取缓冲 t 与表面活性剂 (材料表)。</li>
	<li>储存生物素和磺标签在一个-20 °c 冰箱。 注意: 生物素和磺标签的解决方案应该总是在标签程序前刚准备好。</li>
</ol>2. 用生物素和磺标记人胰岛 Autoantigen
注: 高浓度的抗原, ≥0.5 毫克/毫升, 建议更有效的标签反应。
<ol><li>测定人胰岛 autoantigen 对生物素和磺标记的摩尔比。<ol>
<li>通过分子量除以抗原重, 获得抗原摩尔数。</li>
		<li>使用1:5 的摩尔比的抗原与较小的分子量 (≤10 kd), 和摩尔比率为1:20 的抗原与较大的分子量 (> 50 kd)。</li>
		<li>通过将摩尔数除以浓度, 计算生物素或磺标记的体积。</li>
	</ol>
</li>
	<li>将人胰岛 autoantigen 与生物素或磺标记混合, 摩尔比为1:5。 注: 三种或甘氨酸缓冲系统中的蛋白质应使用 "上浆自旋" 柱, 用 pH 值为7.9 交换到 2x PBS 缓冲器。</li>
	<li>由于生物素和磺标记是轻的敏感, 用铝箔覆盖反应管。在室温下 (RT) 孵育1小时的反应。</li>
	<li>在孵化过程中, 通过将 2x PBS 缓冲区分配到三次柱中, 将自旋柱放在最上方。每一次离心溶液在 1000 x g 2 分钟。 注: 目前的标签反应, 通过衔接, 仍在发生, 需要停止。</li>
	<li>通过纯化标记的人胰岛 autoantigen, 停止标记反应。要净化, 通过 autoantigen 通过旋转柱和离心机的列在 1000 x g 2 分钟。</li>
	<li>用抗原蛋白的数量和最终体积确定标记抗原浓度 (µg/µL)。 注: 大致上, 每一个自旋柱通过后, 将有 90-95% 的标记抗原保留。</li>
	<li>整除标记抗原和存储标记抗原在-80 °c。</li>
</ol>3. 定义检测的两个标记抗原的最佳浓度和比值 (检查板检测)
<ol><li>准备两个血清样本, 一个高阳性和一个阴性, 总容积为每µL 200。</li>
	<li>整除4µL 的血清和添加 1x PBS, 直到最终体积是20µL 每井96井 PCR 板。对高阳性样本使用半盘, 另一半用于负样本, 如图 2A所示。</li>
	<li>添加10µL 的生物素和10µL 的磺标记抗原, 并对两种不同标记的抗原进行串行稀释, 如图 2A所示。对一个标记抗原进行水平序列稀释, 并对其他标记抗原进行垂直序列稀释。</li>
	<li>继续其余的化验步骤, 在5.3 至9.1 所述。</li>
	<li>根据图 2B中显示的阴性样本中的每个对应信号, 计算高阳性样本的信号比。</li>
	<li>通过选择最高或接近阳性率为阴性的点, 确定生物素和磺标记抗原的最佳浓度。考虑从阴性样品的低背景信号, 以确定比例。 注: 化验日1包括步骤4至步骤6。</li>
</ol>4. 用正确浓度的生物素/磺标记抗原制备抗原缓冲器
<ol><li>选择每个抗原的合理浓度的基础上检查板化验。</li>
	<li>用生物素/磺标记抗原的合理浓度对抗原缓冲器进行3毫升抗原溶液的制备。</li>
</ol>5. 用标记抗原孵化血清标本
注: 本节有两种协议, 一是无血清酸治疗, 一是血清酸治疗。除 iaa 检测外, 所有胰岛自身抗体的测定均采用常规的无血清酸治疗方法, 从步骤5.1 到 5.5, 而 iaa 检测跳过这些步骤, 并使用从步骤5.6 到5.9 的血清酸治疗的协议。
<ol><li>整除4µL 的血清和添加 1x PBS, 直到最终体积是20µL 每井96井 PCR 板。</li>
	<li>每井加20µL 标记抗原溶液。</li>
	<li>用密封箔覆盖 PCR 板以避免光线。</li>
	<li>把盘子放在2小时的 RT 上 (低速)。</li>
	<li>把盘子放在4摄氏度的冰箱里, 一夜之间孵化 (18-24 小时)。 注: 以下步骤从5.6 到5.9 的协议仅为 IAA 检测和其他自身抗体化验而设计, 跳过这些步骤。</li>
	<li>将15µL 的血清与18µL 0.5 米醋酸的血清样品混合在一起。在 RT 中孵化这种混合物45分钟。</li>
	<li>采用抗原缓冲剂, 在检测板检测的基础上, 利用生物素和磺标记抗原的合理浓度, 制备抗原溶液。在每一个井中, 整除35µL 抗原缓冲, 含有生物素和磺标记抗原, 进入新的 PCR 板。</li>
	<li>在45分钟孵化 (步骤 5.6) 步骤完成之前, 增加8.3 µL 1 M 三 pH 9.0 缓冲到每个井的边在抗原板材 (步 5.6)。限制三缓冲器与抗原的混合是很重要的。立即转移血清的25µL, 用酸治疗, 在步骤 5.6, 入每个井并且鼓动解答。用密封箔覆盖 PCR 板以避免光线。</li>
	<li>把盘子放在2小时的 RT (低速) 上, 把盘子放在4摄氏度的冰箱里, 让盘子在一夜之间孵化 (18-24 小时)。</li>
</ol>6. 准备链亲和素板
<ol><li>从4摄氏度冰箱中取出一个链亲和素板, 让盘子进入 RT。</li>
	<li>一旦链亲和素板在 RT, 增加150µL 3% 阻滞剂 A 对每个井。</li>
	<li>用密封箔覆盖 PCR 板。</li>
	<li>一夜之间在4摄氏度冰箱里孵化盘子。 注意:化验日2包括步骤7至步骤9。</li>
</ol>7. 将血清/抗原潜伏转移到链亲和素板上
<ol><li>第二天, 从冰箱取出链亲和素板, 从盘子里扔掉缓冲器。把一些干纸巾放在桌子上, 把盘子倒在上面, 直到井内没有缓冲。</li>
	<li>用150µL 的 PBST 填充空链亲和素板, 并将 PBST 从盘子中抹去, 共三洗涤。</li>
	<li>将血清/抗原潜伏的30µL 转移至链亲和素板。</li>
	<li>用箔盖住盘子以避免光线。在一个低的设置, 在 RT 1 小时摇动板。</li>
</ol>8. 洗盘子, 添加读缓冲器
<ol><li>把潜伏从盘子里扔掉。添加150µL 的 PBST 每井, 并丢弃的 PBST 从板块总共三洗涤。</li>
	<li>洗涤后, 增加150µL 每井的读数缓冲区。 注意: 在溶液中防止气泡是很重要的, 因为这会影响板读机上的平板读取方式。</li>
</ol>9. 阅读板和分析数据
<ol><li>数一下板读机上的盘子。抗体值每秒显示为计数 (CPS)。</li>
	<li>将从板读机接收到的抗体水平作为相对指数传递。从以下等式计算索引: 索引值 = [cps (示例)-cps (负控制)]/[cps (正控制)-cps (负控制)]。</li>
	<li>确定阳性和阴性抗体结果使用的截止为阳性, 定义基于第九十九分百分100健康控制对象 (非糖尿病人没有任何已知的自身免疫性疾病和谁没有家庭病史的糖尿病)。</li>
</ol>

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作者没有什么可透露的。

胰岛自身抗体是目前1型糖尿病自身免疫的最可靠的生物标志物。它们标志着胰岛特异性自身免疫的发生, 并确定显性疾病风险。对胰岛自身抗体的 ECL 检测在多国和国际1型糖尿病临床试验中得到了广泛的验证。与目前的 "金" 标准 RBAs 相比, 该检测显示出灵敏度和特异性的提高。通过对高亲和性和高危胰岛自身抗体的鉴别, 通过对澳大利亚央行产生的低风险、低亲和力的信号进行判别, 该方法已显示出其优越的优越性。这在只有单胰岛抗体阳性且从未进展为1型糖尿病的学科中尤为引人注目。这些低亲和性自身抗体的大部分被发现在后续的检测过程中丢失了数月到数年, 表现为 "瞬态阳性"。正如先前假设的, 这些低亲和力 ' 单 ' 抗体可能是由免疫与交叉反应分子。而较高的亲和性, 更高的风险胰岛抗体导致免疫与胰岛抗原本身。此外, ECL 检测已经表明有能力提前的时间, 从目前标准的无线电结合检测 (澳大利亚) 的儿童与糖尿病前, 谁被跟踪到临床糖尿病从出生。一个正在进行的国际临床试验, 糖尿病的环境决定因素, 年轻 (泰迪), 取决于准确的检测, 以确定的时间和外观的第一个胰岛自身抗体 ' 血清 ', 标志着胰岛的开始自身免疫和识别环境诱因。在 ECL 检测中增加灵敏度的一个可能的原因是, 该检测捕获所有免疫球蛋白类: igg、IgM、IgA 或 IgE, 而不是仅依靠检测 igg 的传统的澳洲联储或 ELISA。
在 ECL 化验中, 对于某些特定的自身抗体, 如 IAA, 抗体对抗原的约束力似乎被某些成分在正常的人血清中抑制。为了去除或释放这种抑制, 血清标本的酸性处理是必要的, 在血清的孵化与抗原。如 [图 5] 所示, ECL-IAA 测定法, 对小鼠胰岛素单克隆抗体和患者血清自身抗体与抗原的结合活性有很大的提高。血清样品的酸化, 用于抗体检测, 通常用于解除先前存在的束缚物的分离。在人体血清样品中如何抑制 IAA 信号并通过血清酸化释放的机理尚不清楚, 但此方法已用于其他基于 ECL 的检测14,15。
在一些情况下, 当标记分子, 无论是生物素或磺标记, 抗原蛋白分子的标记 positionsinside 在, 或非常接近抗体结合的关键表位, 这可能会干扰对抗体的结合活动。这将减少化验灵敏度, 并可以彻底撕掉化验。对于常规的标记程序, 每种抗原蛋白分子都需要最大化或饱和度, 通过最大限度地提高每个可能标记位置的标记能力来生成每个标记分子的最大活动或信号。不饱和标记应通过降低标记分子 (生物素和磺标记) 对抗原蛋白的摩尔比, 如果抗原标记成为可能的原因, 抗体结合活动中断。大多数情况下, 主要表位可以更好地保留, 并且可以使用不饱和标记策略释放抗体结合活动的中断。
每种化验都应包括一个内部标准的高正和负控制的指数计算未知样本。在检测的正常控制上限附近的低阳性控制是重要的, 包括监测检测灵敏度。实验室应保持足够整除数的标准阳性和阴性对照, 长期使用, 所有整除数应储存在-20 摄氏度。对于化验质量保证, 化验必须以重复的每一个样本, 每一个阳性结果应确认通过重复样品在一个单独的化验。第三种检测是必要的, 当第二验证性化验不同意第一次化验和两个化验结果, 其中同意 (e. g, +, + 或-,-), 将最终确定一个积极或消极的结果。
通过对单 ECL 检测平台的设置, 可对其进行复用分析。它可以同时确定多达10种不同的自身抗体在一个单井和少量血清。目前, 四个胰岛抗体, 包括 IAA、GADA、IA-2A 和 ZnT8A, 对于 T1D 患者和一般人群的亲属 T1D 进展的风险预测是同等重要的。使用目前单一的自身抗体测量来筛选这4种抗体的方法是费力和低效的, 尤其是大规模的人群筛查。重要的是, 多达40% 的 T1D 患者有额外的自身免疫状况16,17,18。不幸的是, 没有简单和廉价的工具来筛选这些条件。多路 ECL 试验不仅能够将4种目前主要的胰岛抗体检测结合在一起, 而且能够进一步结合其他相关自身免疫性疾病的自身抗体测定。这使得在大规模人群中同时有效地进行多种自身免疫性疾病的高通量筛选成为可能。在多路 ECL 检测中, 如 [图 6] 所示, 在液相形成的每个抗体-抗原复合体将被限制在同一井中的特定链接器源点。车牌读取器上的信号接收器能够识别来自10种不同源点的信号。然而, 具有极高信号的斑点可以产生高的分析背景, 并通过交叉谈话对相邻点造成干扰。因此, 每个抗体检测的上限信号应限制在少于2万的计数。在我们的经验中, 较低背景的自身抗体应放置在相对较远的地方, 在设计现场地图时有较高的计数。对于长期的研究使用多用途的 ECL 检测, 建议使用相同的抗体测定, 以保持该检测一致。

最近建立了一个分期分类系统, 以协助诊断患者 T1D 的初始阶段。准确检测人胰岛自身抗体在识别和分期 presymptomatic 1 型糖尿病中起着重要作用, 因为胰岛自身抗体的存在表明β细胞自身免疫的存在。糖尿病对患者的影响, 从最初发生β细胞自身免疫到症状性疾病, 与胰岛自身抗体的数量和类型有关, 是可变的2,3。
胰岛自身抗体血清、效价和亲和性的年龄会影响症状1型糖尿病的进展率4,5,6,7,8 ,9,10。最近, 已开发的 ECL 检测得到了广泛的验证, 显示了更高的灵敏度, 并有更多的疾病特定的10,11,12,13。这些方法通过早期检测胰岛自身抗体来增强糖尿病风险的预测和分期。它们更准确地标记了胰岛自身免疫的启动, 忽略了与糖尿病无关的低亲和力和低风险信号。
在 ECL 检测中, 血清中的自身抗体, 如果存在, 将磺标记共轭抗原与生物素化捕获抗原结合在液相中。在桥接后, 生物素链接器被捕获在固相中, 并通过 ECL 检测到链亲和素涂层板上的磺标记(图 1)。
本研究主要利用人胰岛抗体的单抗体 ECL 测定。简要地介绍了基于单 ECL 检测的复用抗体检测方法。利用15µL 血清, 多项分析可用于在单井中识别多种、多达10种自身抗体。这种简单的高通量检测方法可用于在一般人群中同时筛选多种相关自身免疫性疾病的多种自身抗体。

<table><tbody><tr><td>Human recombinant proinsulin protein(PINS)</td><td>AmideBio</td><td>Human Proinsulin, Rec</td><td></td></tr><tr><td>Human recombinant GAD65 protein</td><td>Diamyd</td><td>rhGAD65</td><td></td></tr><tr><td>Human recombinant IA-2 protein</td><td>Creative BioMart</td><td>IA2</td><td></td></tr><tr><td>1x Phosphate Buffered Saline (PBS), pH 7.4</td><td>Thermo Fisher Scientific</td><td>10010-023</td><td></td></tr><tr><td>10x PBS, pH 7.4</td><td>Thermo Fisher Scientific</td><td>70011-044</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>SIGMA-ALORICH</td><td>A7906-500G</td><td></td></tr><tr><td>96-well PCR plate&amp;nbsp;</td><td>Fisher</td><td>14230232</td><td></td></tr><tr><td>Streptavidin coated plate</td><td>MSD</td><td>L15SA</td><td></td></tr><tr><td>Zeba sizing spin column</td><td>Thermo Fisher Scientific</td><td>89890</td><td></td></tr><tr><td>Twen-20</td><td>Fisher</td><td>BP337-500</td><td></td></tr><tr><td>HCl</td><td>Fisher</td><td>A144-500</td><td></td></tr><tr><td>NaOH</td><td>Fisher</td><td>SS255-1</td><td></td></tr><tr><td>Trizma Base</td><td>Fisher</td><td>BP152-5</td><td></td></tr><tr><td>EZ-Link Biotin</td><td>Thermo Fisher Scientific</td><td>PI21329</td><td></td></tr><tr><td>Sulfo-tag</td><td>MSD</td><td>R91AO-1</td><td></td></tr><tr><td>Blocker A</td><td>MSD</td><td>R93AA-1</td><td></td></tr><tr><td>4x Read Buffer T with Surfactant</td><td>MSD</td><td>R92TC-1</td><td></td></tr><tr><td>EZ-Link NHS-PEG&lt;sub&gt;4&lt;/sub&gt;-Biotin</td><td>ThermoScience</td><td>21329</td><td></td></tr><tr><td>Sulfo-TAG</td><td>MSD</td><td>R91AO-1</td><td></td></tr><tr><td>96-well polymerase chain reaction (PCR) plate</td><td>Fisher brand</td><td>14230232</td><td></td></tr><tr><td>96-Well Streptavidin plate</td><td>MSD GOLD</td><td>L 15SA-1</td><td></td></tr><tr><td>Zeba Spin Desalting Column</td><td>ThermoScience</td><td>PL208984</td><td></td></tr><tr><td>96-well Plate Shaker</td><td>Perkin Elmer</td><td>1296-003</td><td></td></tr><tr><td>Plate Reader</td><td>MSD</td><td>QuickPlex SQ120</td><td></td></tr><tr><td>Benchtop centrifuge with bucket rotary</td><td>Beckman</td><td>Allegra X-15R</td><td></td></tr></tbody></table>

electrochemiluminescence assays for human islet autoantibodies

精确定位与1型糖尿病 (T1D) 相关的胰岛自身抗体, 在一般人群中导致该疾病的投射和阻吓作用。新的微量液相检测是胰岛自身抗体具有较高的灵敏度和特异性比目前的 "金" 标准无线电结合检测 (澳大利亚)。通过对 RBAs 中产生的低风险、低亲和性自身抗体和常规酶联免疫吸附测定 (ELISA 法) 鉴别高风险、高亲和力的自身抗体, presymptomatic T1D 的发生更精确地定义了其发病。).本试验中使用的抗原蛋白分别标记为磺标记和生物素。每个使用特定抗原蛋白的 ECL 自身抗体检测都需要一个优化步骤才能用于实验室应用。这一步骤对于确定磺标记和生物素两种不同抗原的血清酸处理、浓度和比值的要求尤为重要。在磷酸缓冲液 (PBS) 中, 血清样本与磺标记共轭和生物素化捕获抗原蛋白混合, 含有5% 牛血清白蛋白 (BSA)。之后, 样品在4摄氏度一夜之间孵化。同一天, 链亲和素涂层板准备与阻滞剂缓冲和孵化过夜4摄氏度。第二天, 冲洗链亲和素板, 将血清抗原混合物转移到板上。将盘子放在平板振动筛上, 在低速时设置, 并在室温下孵育1小时。随后, 板再次清洗, 并添加读卡器缓冲区。然后将该板材计入板读机上。结果通过一个指数来传达, 这是由内部标准阳性和阴性对照血清样本产生的。

图 2显示检查器板。结果表明, 250 毫升的生物素和250磺标记抗原是最合理的浓度, 使用的检测信号从高阳性对照样品, 阴性对照样品作为化验的背景, 和比例正向负信号.通过对这两种标记抗原的优化浓度, 对100例新确诊的 T1D 和100健康对照组的血清标本进行了检测。0.023 的指数值被定义为阳性的检测截止。使用图 3A中所示的接收器操作特征 (ROC) 曲线, 这表示患者的85% 敏感度和健康控制中的99% 特异性。用内部标准高阳性、低阳性和阴性对照样品进行了未知样品的检测。CPS 计数的结果显示在表2A 中.高和低正则控制的平均 CPS 突出显示为红色、17903 [(19940 + 15866)/2] 和 839 [(857 + 820)/2], 负控制项以绿色、168 [(170 + 165)/2] 突出显示。未知示例的索引由 "(CPS示例-168)/(17903-168) 计算。表 2B显示所有示例的计算索引值。大于0.023 的索引值用红色编写, 对应于表 2A中也以红色编写的 CPS 值。这些值将被定义为大于健康控制人口的 99th百分比的正结果。当使用不合理的抗原浓度时, 该化验将有较高的背景, 如表 2C所示。在表 2D中以灰色突出显示的值 A2、A5、D1 和 H4 时, 会忽略低电平的阳性抗体。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57227/57227fig1.jpg"> 图 1:在单个 ECL-IAA 检测上的双价板块捕获的图示.血清中的胰岛自身抗体将磺标记共轭抗原与生物素化捕获抗原结合在一起, 在链亲和素涂层板上的固相中捕获。通过 ECL 对磺标记共轭抗原进行检测。此图已从 Yu ( et al) 中修改。11.<a href="/files/ftp_upload/57227/57227fig1large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57227/57227fig2.jpg"> 图 2: 用于确定检测阳性截止度的 ROC 曲线.选择了与85% 灵敏度对应的 99th位数, 并将其表示为0.023 的索引值。这个上限的化验是从100健康的控制。<a href="/files/ftp_upload/57227/57227fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57227/57227fig3.jpg"> 图 3:正常人血清阻断 ECL-IAA 信号的图示.A: 用胰岛素单克隆抗体 (摩押) 检测的来自 ECL-IAA 检测的信号被添加正常的人血清严重阻断. 比较在 PBS 中的摩押时, 当人血清用酸治疗时, 信号部分恢复。B: 用酸性疗法显著提高4例血清中的 ECL-IAA 检测信号。此图已从 Yu ( et al) 中修改。11.<a href="/files/ftp_upload/57227/57227fig3large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57227/57227fig4.jpg"> 图 4:多重 ECL 检测的说明.抗体-抗原复合物在液相与特定连接器形成。在同一井中, 特定的抗体-抗原-链接器复合物在每个特定的连结点上都受到限制。板读机能够区分不同源点的信号, 分别给出10种不同信道的 CPS 计数。<a href="/files/ftp_upload/57227/57227fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/57227/57227tbl1.jpg"> 表 1: 检查器板检测确定生物素和磺标记抗原的浓度和比值.A: 原始 CPS 计数为检查板板与高阳性血清在一半的板材和阴性血清在另一半。生物素化抗原浓度在级数上呈水平稀释, 磺标记抗原浓度呈垂直稀释。B: cps 的比值值从阴性血清中的每个对应 cps 计数的高阳性血清计数。在 B 面板中, 黄色突出的井代表最佳的阳性比率。这个比例对应于 250 ng/毫升生物素和250磺标记抗原浓度在面板 A 与可接受的低 CPS 计数阴性血清。<a href="/files/ftp_upload/57227/57227tbl1large.jpg" target="_blank">请单击此处查看此表的较大版本.</a>
<img alt="Table 2" src="/files/ftp_upload/57227/57227tbl2.jpg"> 表 2: 化验结果分析.A: 原始 CPS 计数从化验盘与标准高和低正面控制突出红色和标准阴性控制突出显示在绿色。每个样品在化验中重复。B: 计算指数值, 如化验协议所述。任何大于0.023 的索引值都被定义为正结果, 以红色突出显示。C: 在使用非理性抗原浓度时, 原始 CPS 从检测盘中计数。这导致了较高的背景和一些通常检测到的低阳性, 如面板 B 所示, 被转换为底片, 在面板 D 中以灰色突出显示.<a href="/files/ftp_upload/57227/57227tbl2large.jpg" target="_blank">请单击此处查看此表的较大版本.</a>

Watch this Scientific Journal Video about Electrochemiluminescence Assays for Human Islet Autoantibodies at JoVE.com

Electrochemiluminescence Assays for Human Islet Autoantibodies

在这里, 我们提出了检测人胰岛抗体的方法, 使用电化学发光 (ECL) 测定。该协议, 用于预测1型糖尿病, 可以扩大, 以检测自身抗体的其他自身免疫性疾病。

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 ...

electrochemiluminescence-assays-for-human-islet-autoantibodies

Research

JoVE Journal

Immunology and Infection

15.4K 次观看.  University of Colorado Denver. 在这里, 我们提出了检测人胰岛抗体的方法, 使用电化学发光 (ECL) 测定。该协议, 用于预测1型糖尿病, 可以扩大, 以检测自身抗体的其他自身免疫性疾病。

视频: Electrochemiluminescence Assays for Human Islet Autoantibodies

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

MeCP2蛋白质变异的电化学发光测定

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

科研

生物化学

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 ...

免疫与感染

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Monospecific 和混合 DFS70 模式在 HEp-2 ELITE/DFS70 击倒底物筛选中的同时鉴别

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...

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 ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

提取组织抗原的功能分析

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

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 ...

医学

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

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 ...

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

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.