Name: a sensitive and specific quantitation method for determination of serum cardiac myosin binding protein-c by electrochemiluminescence immunoassay
Uploaded: 2013-08-08T11:00:00
Description: Watch this Scientific Journal Video about A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay at JoVE.com

资助这项研究是由美国国立卫生赠款R01HL105826 K02HL114749（博士Sadayappan），美国心脏协会中西部奖学金; 11PRE7240022（先生Barefield）和13POST14720024（Govindan博士）。作者非常感谢其出色的技术和文献支持在开发检测的协助下，吉尔Clampit的吉米·佩奇博士和约翰·哈德森博士，MSD。

1。 Uniplex cMyBP C含量<ol><li>前一天实验，大衣96以及MSD裸的标准板捕获抗体。对于-C cMyBP，使用的浓度为5微克/毫升稀释于磷酸盐缓冲盐水（PBS）30μl的体积的小鼠单克隆抗cMyBP-C抗体（明胶免费）。 </li><li>由于是疏水性的，吸液管的解决方案以及入下角，然后点击两侧的板块，传播的解决方案，在整个井。 </li><li>板封口板覆盖着，在4°C孵育O / N无晃动。 </li><li>卸下捕获抗体溶液，攻解掉在水槽上...

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在这项研究中，我们表明多个心脏生物标记物的检测患者血清中的多重免疫测定法的适用性。这种技术有许多优点，比传统的ELISA。首先，使用ECL检测试剂盒中​​的，而不是比色检测。在ECL，电信号刺激当地生产的测量属性（光），从而解偶联信号的刺激事件，从而降低背景14。这允许高灵敏度，可以看出在表2中。 复用不会导致检测的敏感度的降低，?...

低量的蛋白质，在复杂生物样品，如血清，测量病人的管理，以及基础科​​学发展的临床重要性。比如，增加血清心脏生物标志物，如肌钙蛋白I，在临床的设置是一致的急性心肌梗死（MI）1。为了检测血清样品中的蛋白质，标准的酶联免疫吸附试验（ELISA）法是最常用的技术，因为它具有高灵敏度和允许的被分析物的绝对定量。然而，传统的ELISA试剂盒，需要一个比较大的量的样品（一般为100微升），在生物体液中的一些具有高的背景信号，并且被限制为只有一个每2酶联免疫吸附分析物的测量。 最近，一种新的免疫分析技术被引入的情况下，许多这些缺点。此修改后的实验中，由MSD的开发，使用electrochemiluminescENCE（ECL）信号的检测，允许一个非常低的背景和敏感性的增加，能够使用小样本量。电致化学发光是根据记者分子钌（II）trisbipyridal，这是附加的检测抗体。记者分子发射光在620nm时的底部的96 -孔的板，它具有集成在其中3个碳电极的电刺激。另外，通过使用点涂，多个捕获抗体可以被涂覆到一个阱（最多10个96孔板上），允许单个样品中4种不同的蛋白质的同时定量。最近这种技术已被用来衡量促炎细胞因子的血清5,6。从MSD多重板媲美其他多重检测平台7。 我们使用MSD作为主要的试验平台，进一步开发一个定制的复杂板的C同时量化心肌肌球蛋白结合蛋白C（cMyBP-C），肌酸激酶同工酶（CK-MB），心肌肌钙蛋白I（cTnI）的水平进行比较，其结果与monoplex检测的cMyBP C。 CK-MB和cTnI MI行之有效的生物标志物。然而，这些生物标志物的增加可引起心肌梗死以外的其他病症， 如心肌炎或肾功能衰竭8。认为除了增加额外的生物标志物的特异性心肌梗死的诊断。最近，我们已经表明cMyBP-C也是一个潜在的生物标志物MI 9。 cMyBP-C是一种粗丝相关蛋白表达在心脏，10-12，但不是在肌肉骨骼或光滑。因此，循环系统中的cMyBP-C的水平的提高是一个特定的心脏损伤的指标13。 在这项研究中，我们比较了使用自定义复杂的检测与测量血清的cMyBP C uniplex检测，CK-MB cMyBP-C和在MI患者血清肌钙蛋白I。在未来，这种签名技术可以用来诊断心肌梗死的患者，在急诊室出现胸痛。 美国芝加哥Loyola大学的机构审查委员会（IRB）批准了这项研究中使用的人类deidentified样本和使用的免疫法（LU＃20392）。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>MSD Read-Buffer T (4X) with surfactant</td>
			<td>Meso Scale Discovery</td>
			<td>R92TC-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Acros</td>
			<td>9005-64-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MSD Blocker A</td>
			<td>Meso Scale Discovery</td>
			<td>R93BA-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10X</td>
			<td>Fisher BioReagents</td>
			<td>BP399-4</td>
			<td>Filter before use</td>
		</tr>
		<tr>
			<td>Myosin binding protein C antibody, mouse monoclonal (E7), gelatin free</td>
			<td>Santa Cruz</td>
			<td>SC-137180L</td>
			<td>For coating, antibody has to be gelatin free</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Plates and Equipment</td>
		</tr>
		<tr>
			<td>Multi-Array 96-well standard plate</td>
			<td>Meso Scale Discovery</td>
			<td>L15XA-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A prototype 3-plex, four-spot, 96-well plate with cMyBP-C, cTnI and CK-MB</td>
			<td>Meso Scale Discovery</td>
			<td>N452A-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ThermaSeal Sealing Film</td>
			<td>Life Science Products Inc.</td>
			<td>ST-3098</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MSD SECTOR Imager 2400A</td>
			<td>Meso Scale Discovery</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MTS 2/4 digital microtiter shaker</td>
			<td>IKA</td>
			<td>3208001</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a sensitive and specific quantitation method for determination of serum cardiac myosin binding protein-c by electrochemiluminescence immunoassay

生物标志物正变得越来越重要，在临床决策，以及基本的科学。诊断心肌梗死（MI）主要是由患者的血清或血浆中检测心肌特异性蛋白作为心肌损伤的指标。最近的研究表明心肌肌球蛋白结合蛋白C（cMyBP-C）MI后的血清中检测到，我们提出了它作为一种潜在的生物标志物的MI。生物标志物检测通常由传统夹心酶联免疫吸附试验。然而，该技术需要一个大的样品体积，有一个小的动态范围，并且可以测量在一个时间只有一个蛋白质。 在这里，我们示出了复用的免疫测定法，它可以同时测量三个心脏的蛋白质具有很高的灵敏度。测量cMyBP-C在uniplex或共同肌酸激酶MB和肌钙蛋白，我发现类似的敏感性。这种技术使用细观发现（MSD）的复用的方法，在96 - 孔的板，用于检测电致化学发光结合。虽然只有少量样品的要求，实现高灵敏度和大动态范围。使用这种技术，我们测量cMyBP-C，肌酸激酶MB，心肌肌钙蛋白I血清样品从16个科目与MI水平，并与16个对照组比较的结果。我们能够检测这些样品中的所有三个标记和发现所有三种生物标志物来增加心肌梗死后。因此，这种技术是适用于敏感的检测血清样品中的心脏生物标记物的。

3复杂的检测的基本原理和工作流程示于图1，表1中记载的总体工作流程。 uniplex检测沿相同的原理工作的整个底部与捕获抗体以及涂覆除外。信号的检测是通过ECL的电信号被施加到井的底部。这反过来SULFO-TAG标签检测抗体的读缓冲区通过化学反应，将启动一个本地生产的光。这导致了低背景和高灵敏度的检测。在图2中的cMyBP-C在uniplex检测的标准曲线比较的cMyBP中的C 3-plex?...

Watch this Scientific Journal Video about A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay at JoVE.com

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

测量越来越复杂的生物样品中的生物标志物指导临床决策。我们描述一个高度敏感的方法同时测量心肌肌球蛋白结合蛋白C，肌酸激酶MB，心肌肌钙蛋白I与心肌梗死和健康对照组受试者的血清样本。

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

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