Name: conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method
Uploaded: 2012-07-06T14:00:00
Duration: 14 min 48 s
Description: Watch this Scientific Journal Video about Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method at JoVE.com

这项工作是由Luminex公司。

<ol>
	<li>Fulton, J. R., McDade, R. L., Smith, P. L., Kienker, L. J., Kettman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+multiplexed+analysis+with+the+FlowMetrix+system.">Advanced multiplexed analysis with the FlowMetrix system.</a> Clinical Chemistry. 43, 1749-1756 (1997).</li><li>Carson, R. T., Vignali, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantation+of+15+cytokines+using+a+multiplexed+flow+cytometric+assay.">Simultaneous quantation of 15 cytokines using a multiplexed flow cytometric assay.</a> J. Immunol. Methods. 227, 41-52 (1999).</li><li>Peck, D., Crawford, E. D., Ross, K. N., Stegmaier, K., Golub, T. 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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+extract+cytokines+and+matrix+metalloproteinases+from+Schirmer+strips+and+analyze+using+Luminex.">A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex.</a> Mol Vis. 17, 1056-1063 (2011).</li><li>Liu, J., Kibiki, G., Maro, V., Maro, A., Kumburu, H., Swai, N., Taniuchi, M., Gratz, J., Toney, D., Kang, G., Houpt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+reverse+transcription+PCR+Luminex+assay+for+detection+and+quantitation+of+viral+agents+of+gastroenteritis.">Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis.</a> J. Clin. Virol. 50, 308-313 (2011).</li><li>de Jager, W., Prakken, B. J., Bijlsma, J., WJ Kuis, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+multiplex+immunoassay+performance+in+human+plasma+and+synovial+fluid+following+removal+of+interfering+heterophilic+antibodies.">Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.</a> J. Immunol. Methods. 300, 124-135 (2005).</li><li>Codorean, E., Nichita, C., Albulescu, L., Raducan, E., Popescu, I. D., Lonita, A. C., Albulescu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+xMAP+and+ELISA+cytokine+profiles;+development+and+validation+for+immunotoxicological+studies+in+vitro.">Correlation of xMAP and ELISA cytokine profiles; development and validation for immunotoxicological studies in vitro.</a> Roum. Arch. Microbiol. Immunol. 69, 3-19 (2010).</li><li>de Jager, W., te Velthuis, H., Prakken, B. J., Kuis, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+15+human+cytokines+in+a+single+sample+of+stimulated+peripheral+blood+mononuclear+cells.">Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.</a> Clin. Diagn. Lab. Immunol. 10, 133-139 (2003).</li><li>DuPont, N. C., Wang, K. H., Wadhwa, P. D., Culhane, J. F., Nelson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+comparison+of+Luminex+multiplex+cytokine+analysis+kits+with+ELISA:+Determinations+of+a+panel+of+nine+cytokines+in+clinical+sample+culture+supernatants.">Validation and comparison of Luminex multiplex cytokine analysis kits with ELISA: Determinations of a panel of nine cytokines in clinical sample culture supernatants.</a> J. Reprod. Immunol. 66, 175-191 (2005).</li><li>Richens, J. L., Urbanowicz, R. A., Metcalf, R., Corne, J., O'Shea, P., Fairclough, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+validation+and+comparison+of+multiplex+cytokine+kits.">Quantitative validation and comparison of multiplex cytokine kits.</a> Journal of Biomolecular Screening. 15, 562-568 (2010).</li><li>Rizzi, G., Zhang, Y. J., Latek, R., Weiner, R., Rhyne, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+development+of+a+Luminex-based+assay+for+the+detection+of+human+IL-23.">Characterization and development of a Luminex-based assay for the detection of human IL-23.</a> Bioanalysis. 2, 1561-1572 (2010).</li></ol>

这项工作是在Luminex公司与Luminex公司制造的设备。 研发系统与EMD的微孔Luminex公司的战略合作伙伴;许可的开发和商品化基于多重XMAP的检测。

酶联免疫吸附试验转换的Luminex XMAP平台，可以简单，只要代链霉亲和素辣根过氧化物酶（SA-HRP）的，在一个典型的链霉，藻红蛋白（SA-PE）的ELISA试剂盒和性能优化。对于那些谁想要建立XMAP免疫，从地上爬起来，这可以用一个简单的协议，也使快速，多重抗体对评价完成。为XMAP检测试剂很容易被准备使用XMAP抗体偶联试剂盒夫妇指定的捕获抗体MagPlex低浓度微。使用低浓度微减少，而高浓度微提供相同的检测性能检测方法开发的成本。的准备再加MagPlex微所需的时间量是大约3个小时，这是速度远远超过了22 - 24小时大衣以及酶标板治疗涂井。的XMAP法的性能也优于酶联免疫吸附检测（&lt;4皮克/毫升与31皮克/毫升）和动态范围（&lt;4皮克/毫升至8000皮克/毫升比16的限制条款皮克/毫升至1000皮克/毫升）。板读者有一个有限的外径范围是3或4外径，限制检测的动态范围为上限。 毫无疑问，并非所有的抗体，将工作在ELISA格式，并非所有的抗体，在酶联免疫吸附试验工作，随时转让的XMAP检测格式。然而，因为XMAP检测可复用（例如，同时运行），它是可能的，以评估数的捕获和检测抗体组合，同时使用一个试验以确定最佳的一双。这个过程中可以节省大量的时间和试剂相比，酶联免疫吸附试验发展过程中，时间是有限的一对评价。如果两个或两个以上的抗体对执行等价;其他参数的检测被认为是对的（例如，可用性，成本等），以确定适宜。 除了与XMAP检测以提高​​检测性能和灵活性，也有显着的成本节约。建议的抗体数量所需的外套单井酶标板是400ng，而用于一个XMAP检测珠所需的数量大约是7.5纳克。因此，所需金额为酶联免疫吸附试验的抗体，将提供超过50个测试结果，如果用在XMAP法。对于涉及珍贵样品的应用，XMAP也有一个显着的优势。建议的酶联免疫吸附试验的样本量为100μL，而为XMAP检测所需的数量可以是一半，或更少。 总之，酶联免疫吸附试验的Luminex XMAP平台转换是简单，高效，节约成本，同时生产具有优异的动态范围和灵敏度的检测。

<table border="1"><tbody><tr><td> 描述 </td><td> 供应商 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>人类TNF-α/TNFSF1ADuoSet ELISA试剂盒</td><td> R＆D系统</td><td> DY210 </td><td>包括单克隆抗体和生物素耦合多克隆抗体和重组肿瘤坏死因子-α蛋白标准</td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> abcam </td><td> ab18696 </td><td>捕获抗体，克隆CH8820 </td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> abcam </td><td> ab16166 </td><td>生物素耦合检测抗体，克隆AS1的</td></tr><tr><td>肿瘤坏死因子α蛋白</td><td> abcam </td><td> ab9642 </td><td>重组肿瘤坏死因子-α蛋白标准</td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> ñovus </td><td> NBP1-50115 </td><td>捕获抗体，克隆4H31 </td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> Novus公司</td><td> NB100-78162 </td><td>生物素耦合检测抗体，克隆MAb11 </td></tr><tr><td>肿瘤坏死因子α蛋白</td><td> Novus公司</td><td> NBC1-18460 </td><td>重组肿瘤坏死因子-α蛋白标准</td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> EMD的微孔</td><td> MAB1141 </td><td>捕获抗体，克隆3C7.2 </td></tr><tr><td>肿瘤坏死因子-α多克隆抗体</td><td> EMD的微孔</td><td> 654250 </td><td>兔多克隆抗体检测抗体</td></tr><tr><td>链霉亲和素 - 藻红蛋白</td><td>苔</td><td> SAPE-001 </td><td>荧光记者点阵仪XMAP含量的试剂</td></tr><tr><td> MAGPIX W / xPONENT软件</td><td> Luminex公司</td><td>马GPIX-XPONENT </td><td> Luminex液相仪器</td></tr><tr><td> XMAP抗体偶联（ABC）套件</td><td> Luminex公司</td><td> 40-50016 </td><td>包括EDC试剂，磺酸基-NHS的试剂，激活缓冲，洗涤缓冲液，1.5 ml反应管，一次性移液器</td></tr><tr><td> MagPlex微球，低浓度</td><td> Luminex公司</td><td> MC10012-ID，MC10013-ID，MC10014-ID，MC10015编号</td><td>低浓度珠（@ 2.5×10 6珠/毫升） </td></tr><tr><td> EZ-Link的磺酸基-NHS的 - LC-生物素</td><td>赛默飞世尔</td><td> PI-21335 </td><td>未修改检测抗体生物素试剂盒</td></tr><tr><td> Tecan公司无限F200的读者</td><td> Tecan公司</td><td></td><td>酶标板读卡器</td></tr><tr><td>磷酸盐缓冲液</td><td> Sigma-Aldrich公司</td><td>的P-3688 </td><td> 1％PBS-BSA的，缓冲液; </td></tr><tr><td>一品脱的紧凑型超声波清洗机，115 VAC </td><td>科尔 - 帕默</td><td>吴-08849-00 </td><td>产生有效工作频率为55千赫</td></tr><tr><td>磁管分离器</td><td> Luminex公司</td><td> CN-0288-01 </td><td>对于单一1.5ml离心管中磁分离耦合的洗涤步骤</td></tr><tr><td>磁板分离器</td><td> Luminex公司</td><td> CN-0269-01 </td><td>对于96孔板磁分离检测洗涤步骤</td></tr></tbody></table>

conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method

酶联免疫吸附试验（ELISA）长期以来的利益为生命科学研究和临床诊断的生物样品的分析物检测的主要工具。然而，酶联免疫吸附试验有其局限性。它通常在96孔板，井都涂有捕获抗体，需要的样本量相对较大，捕获抗原的利益。表面积大井和捕获抗体的疏水约束力，也可以导致非特异性结合和增加的背景。此外，大多数的ELISA依靠酶介导的信号放大，以达到合理的灵敏度。这种放大是不总是线性的和可倾斜的结果。 在过去的15年中，出现了一种新的技术，提供了酶联免疫吸附试验的好处，但也实现更高的吞吐量，增加灵活性，减少样本量，降低成本，具有类似禾rkflow 1，2。 Luminex液相xMAP技术是1（珠）微阵列平台，使双方monoplex和复用，可应用于蛋白质和核酸的应用3-5检测。珠捕获抗体共价固定在一个较小的表面积，需要较少的捕获抗体和样本量较小，比较酶联免疫吸附试验，非特异性结合是显著减少。与限制样品，如脑脊液，滑液等6个工作时，较小的样本量是重要的。复检测，进一步降低样本量的要求，使多个结果，从一个单一的样本。 最近由点阵仪的改进包括：新MAGPIX系统，体积更小，更便宜，更易于使用分析仪;低浓度MagPlex磁性微球的消除需要昂贵的过滤板，来更好的检测方法开发适合的工作浓度和低吞吐量应用; XMAP抗体偶联（ABC）的工具包，其中包括一项协议，试剂，消耗品耦合珠捕获抗体的利益所必需的。 （见材料第一个包内容的详细清单。） 在这个实验中，我们的XMAP平台转换为TNF-α的细胞因子的预优化的酶联免疫吸附试验和比较两种方法的性能7-11。 TNF-α是一种生物标志物用于测量与自身免疫性疾病患者的炎症反应。 我们开始由四个不同的微套或地区耦合四个候选人捕获抗体。当混合在一起，这四套允许有四个单独的抗体检测方法的所有四名候选人，以确定最佳的抗体对，节省试剂，样品和时间同步测试。 ，两个XMAP检测构建两个最优化的抗体对他们的表现相比原ELISA法检测到的信号强度，动态范围和灵敏度方面。

Watch this Scientific Journal Video about Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method at JoVE.com

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

ELISA方法可以很容易地转换到一个点阵仪XMAP法，通过复用，几种抗体的好处，可以同时进行筛选，以确定最佳的抗体对，导致更高的灵敏度和动态范围，同时降低检测成本。

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 ...

Research

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Biology

Laser Capture Microdissection of Mammalian Tissue

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

科研

生物学

Cell Capture Using a Microfluidic Device

细胞捕获使用一个微流体装置

Method for Whole Mount Antibody Staining in Chick

整装在鸡的抗体染色方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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

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 ...

Anti-Nuclear Antibody Screening Using HEp-2 Cells

抗核抗体筛选使用Hep-2细胞

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...