国立卫生研究院资助RC1-HL-101102，P01-CA039542，T32-HL007622，哈特韦尔基金会，和多丽丝公爵慈善基金会的支持下。 Paczesny博士是研究员埃里克·哈特韦尔基金和艾米Strelzer的Manasevit研究计划。

1。实验第0天：样品制备和ELISA检测涂层钢板捕获抗体，IL-2Rα，REG3α和肝细胞生长因子<ol><li>血浆的等分试样以进行分析样品将被拉到，解冻，并在12,000 rpm离心10分钟以分离在底部和顶部从血浆中的脂质对血栓纺丝。到一个96孔V底板上，从每个样品中加入150μl未稀释的等离子体将被镀（源板）由手动移液，根据预先定义的映射。等分试样将在封口膜包裹，并在整个过程中保持在潮湿的腔室中，在4℃，不长于72小时。 </li><li> IL-2Rα和HGF捕获抗体将会重组，每个制造商的规格和50μl稀释成各自的96孔高结合半孔板中，然后将其密封，并温育过夜，在4℃下的各孔镀覆另外，许多板，可以在37℃下干燥，并保存在4℃，供以后使用，取决于ING上的蛋白质的稳定性。 </li><li>将根据制造商的协议，使用制造商的包被缓冲液和25μl稀释REG3α捕获抗体将被镀在384孔Nunc公司马克西SORP板的各孔中，然后密封，并温育过夜，在4℃下</li></ol> 2。实验第1天：IL-2Rα的酶联免疫吸附（图1） <ol><li>洗涤的IL-2Rα的试验板，在TBS与BLOTTO中断，标准重组，一个8点的标准曲线制备按制造商协议。 </li><li>洗涤后的板的分块步骤之后，加入50μl未稀释的血浆镀在重复从源板的ELISA试验板，和50μl的每个标准镀一式两份。板密封并在室温下孵育2小时，在平板上旋转器设定为300rpm。 </li><li>回收等离子体从IL-2Rα的酶联免疫吸附试验板，并重新设置在未稀释等离子体源板。 ELISA完成每个制造商协议（与调整一半孔板的卷）的各孔的光密度，使用读板器设置为450-570纳米，将被读取，并且数据保存和分析。 </li></ol> 3。实验第1天：REG3αELISA（图1） <ol><li>将被转移到一个单独的V形底部源板10微升未稀释的血浆。制造商提供的稀释缓冲液的90微升被加入到各孔中，以创建一个1:10稀释源板。 </li><li> REG3αELISA法进行每个制造商的协议（384孔板调整与卷）和各孔的光密度值，将被设置为450-620 nm的用酶标仪读取，并且将被保存的数据和分析。 </li></ol> 4。实验第1天：Elafin和的TNFR1测试涂层钢板捕获抗体<ol><li> Elafin和TNFR1捕获抗体的重组和稀uted每个制造商的规格和50μl接种到各自的96孔高结合半孔板中，然后将其密封，并在室温下温育过夜Elafin的各孔中，并在4℃下TNFR1。 </li></ol> 5。实验日1-2：HGF ELISA（图1） <ol><li>完成后IL-2RαELISA和保证测试并不需要重复，60微升未稀释的血浆将转移到一个新的源板，然后被加入到各孔60微升1×PBS的1％BSA的作出1:2稀释血浆源板。 </li><li>洗涤HGF的试验板，在TBS与BLOTTO中断，标准重组，一个8点的标准曲线制备按制造商协议。 </li><li>洗涤后的板的分块步骤之后，加入50μl的1:2稀释血浆镀上的酶联免疫吸附试验板，一式两份，每一个标准的50微升镀一式两份。该板被密封并在室温下温育过夜，在平板上旋转器设置在300 RPM。 </li><li> 1:2稀释血浆肝细胞生长因子酶联免疫吸附试验板被收回，并放置在1:2的稀释血浆源板。 ELISA完成每个制造商协议（与调整一半孔板的卷）的各孔的光密度，使用读板器设置为450-570纳米，将被读取，并且数据保存和分析。 </li></ol> 6。实验第2天：Elafin ELISA <ol><li>将被转移到一个新的源板10微升未稀释的血浆，然后在1×PBS中的1％BSA的190微升将被添加到每个孔中，使200微升血浆dluted 1:20。 </li><li> Elafin ELISA法，根据制造商的协议（与卷调整半孔板），以及各孔的光密度，将被设置为450-570 nm的使用平板读取器读出，并保存的数据和分析。 </li></ol> 7。实验大Y 2：TNFR1 ELISA <ol><li>将被添加到1:20源板（现在1:20等离子体含有100微升），得到125微升1:25稀释血浆25μl的1％BSA的PBS。 </li><li> TNFR1 ELISA的每个制造商协议完成（与调整一半孔板的卷）和各孔的光密度，使用读板器设置为450-570纳米，将被读取，并保存的数据和分析。 </li></ol> 8。实验第2天：IL-8 ELISA <ol><li> 1:2稀释血浆的60微升转移到一个新的源板，然后被添加到各孔中，使一个1:6的稀释血浆源板180μl的IL-8的稀释剂。 </li><li>的IL-8 ELISA的每个制造商协议完成（与调整一半孔板的卷）和将被读取各孔的光密度值，使用平板读取器设置为450-570纳米，和保存的数据和分析。 </li></ol>一旦所有的ELISA试剂盒已经完成，unuseD存量等离子体将被替换成解冻的等分试样，并冷冻，以备将来使用。

<ol>
	<li>Paczesny, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomarker+panel+for+acute+graft-versus-host+disease.">A biomarker panel for acute graft-versus-host disease.</a> Blood. 113, 273-278 (2009).</li><li>Paczesny, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elafin+is+a+biomarker+of+graft-versus-host+disease+of+the+skin.">Elafin is a biomarker of graft-versus-host disease of the skin.</a> Science Translational Medicine. 2, 13ra12 (2010).</li><li>Ferrara, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+islet-derived+3+alpha+is+a+biomarker+of+gastrointestinal+graft-versus-host+disease.">Regenerating islet-derived 3 alpha is a biomarker of gastrointestinal graft-versus-host disease.</a> Blood. , (2011).</li><li>Ferrara, J. L., Levine, J. E., Reddy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft-versus-host+disease.">Graft-versus-host disease.</a> Lancet. 373, 1550-1561 (2009).</li><li>Miyamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+concentration+of+the+soluble+interleukin-2+receptor+for+monitoring+acute+graft-versus-host+disease.">Serum concentration of the soluble interleukin-2 receptor for monitoring acute graft-versus-host disease.</a> Bone Marrow Transpl. 17, 185-190 (1996).</li><li>Holler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+tumor+necrosis+factor+alpha+in+acute+graft-versus-host+disease+and+complications+following+allogeneic+bone+marrow+transplantation.">Role of tumor necrosis factor alpha in acute graft-versus-host disease and complications following allogeneic bone marrow transplantation.</a> Transplant. Proc. 25, 1234-1236 (1993).</li><li>Okamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+hepatocyte+growth+factor+in+serum+in+acute+graft-versus-host+disease.">Increased hepatocyte growth factor in serum in acute graft-versus-host disease.</a> Bone Marrow Transpl. 28, 197-200 (2001).</li><li>Uguccioni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+interleukin-8+serum+concentrations+in+beta-thalassemia+and+graft-versus-host+disease.">Elevated interleukin-8 serum concentrations in beta-thalassemia and graft-versus-host disease.</a> Blood. 81, 2252-2256 (1993).</li><li>Osuchowski, M. F., Siddiqui, J., Copeland, S., Remick, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+ELISA+to+profile+multiple+cytokines+from+small+volumes.">Sequential ELISA to profile multiple cytokines from small volumes.</a> J. Immunol. Methods. 302, 172-181 (2005).</li><li>Osuchowski, M. F., Remick, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repetitive+use+of+samples+to+measure+multiple+cytokines:+the+sequential+ELISA.">The repetitive use of samples to measure multiple cytokines: the sequential ELISA.</a> Methods. 38, 304-311 (2006).</li><li>Schweitzer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+profiling+on+microarrays+by+rolling-circle+amplification.">Multiplexed protein profiling on microarrays by rolling-circle amplification.</a> Nat. Biotechnol. 20, 359-365 (2002).</li></ol>

没有利益冲突的声明。

<html>顺序ELISA这里介绍的方法可以用于测量多个血浆蛋白，这可能是难以获取和/或不可替代的，如从人类受试者的样品与罕见疾病或血浆样品从小鼠9,10获得关于小体积的血浆。通常执行的顺序的ELISA在增加血浆稀释因子的顺序，用ELISA试剂盒需要血浆稀释的≥1:10通常不需要被回收的，虽然如果需要的话，可以做到。的能力来执行顺序的酶联免疫吸附由等离子体与其它试剂混合，或为不同的稀释液所需要的等离子体的ELISA试剂盒/协议的限制，这排除了重新使用一个样本ablility由于担心不兼容的缓冲/试剂会干扰与一个特定的测试的性能。通过精心规划，10个或更多的ELISA试剂盒可以执行相同的血浆样本。 ENT“&gt;个人实验室可能需要调整血浆稀释，以便有根据预期的血药浓度的蛋白质样品从考试科目的兴趣可解释的结果。实验室设备的差异，可能会导致在需要优化的孵化和色度发展次，洗涤次数和/或洗浸泡时间，以优化任何给定的ELISA法。 为了增加高通量能力和准确性，在成本效益的方式进行分析，使用机器人的液体处理平台，能够分析在384孔板上堆积单元和一个自动洗板推荐。该设备可以提高准确度和精密度由多个用户进行分析，并提供分析的一致性，以减少间和批内变异。 我们使用顺序ELISA的可复用平台的原因有两个：1）大部分的新型蛋白质的抗体对不能很容易地珠或其它材料上，以及共轭耗时和昂贵的; 2）单独的ELISA测定是更精确的比复用微阵列或珠粒，继发没有交叉反应性11。如果建立了一种可靠的方法来执行多路复用，珠为基础的微阵列，它可以是能够替换的顺序ELISA过程，但可能是有限的共轭的抗体的能力，珠和/或期望的蛋白质由数量进行了分析。

3个器官系统功能障碍：急性移植物抗宿主病（GVHD），异基因造血干细胞移植（HSCT）的非复发死亡率（NRM）的首要原因，是衡量皮肤，肝脏和胃肠道（GI）道4。急性移植物抗宿主病通常发生移植后2至8个星期，但以后可能会出现，往往是临床上区分其他造血干细胞移植后的并发症，如预处理方案的毒性，感染或药物副作用。通过使用蛋白质组策略和高通量的验证，使用连续的ELISA，我们已经确定了6种蛋白质的浓度升高开始时，移植物抗宿主病的临床表现。 IL-2Rα，TNFR1，HGF和IL-8，组合成4的生物标志物面板时，可诊断移植物抗宿主病发病的临床症状，并能独立预测造血干细胞移植后生存期的移植物抗宿主病（GVHD）的严重性1。 Elafin，移植物抗宿主病的生物标志物的SK，可以区分移植物抗宿主病（GVHD）的皮疹和疹因其他原因（如药疹）和可以预测移植存活2。我们最近确定REG3α的作为生物标志物，移植物抗宿主病的下消化道，靶器官与NRM。的等离子REG3α浓度能够可靠地识别移植物抗宿主病造血干细胞移植后腹泻的原因和相关的诊断肠道活检组织学，移植物抗宿主病的严重程度。胃肠道移植物抗宿主病发病REG3α浓度也可以预测移植物抗宿主病治疗和NRM 3的响应。将这些经过验证的移植物抗宿主病（GVHD）的生物标志物进入临床护理临床试验，目前正在调查中。 这些实验是在接受造血干细胞移植治疗的患者，在2000年至2010年间的移植物抗宿主病（GVHD）发生的时间，是不可替代的，数量有限的小收集的血浆等份。由于这些样本的珍贵性，我们已经开发出一种MET排泥测量多个血浆蛋白浓度，在一个有效的，可重复的方式，以消除过量的冻结 - 解冻周期，解冻时间和血浆使用。此技术也可应用于在同一样品上执行多个感兴趣的蛋白/细胞因子ELISA试剂盒（）提供的样品不需要与其它试剂混合。如果ELISA试剂盒不配备预涂覆板，96孔半孔板或384孔板可以使用，以进一步减少使用的样本/试剂。稿件侧重于技术方面的测量移植物抗宿主病（GVHD）的生物标志物。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>人类IL-2 R字母DUOSET </td><td> R＆D Systems公司</td><td> DY223 </td><td></td></tr><tr><td>人肝细胞生长因子DUOSET </td><td> R＆D Systems公司</td><td> DY294 </td><td></td></tr><tr><td>人类IL-8 OptEIA KIT II </td><td> Becton Dickinson公司</td><td> 550999 </td><td></td></tr><tr><td> AB匹配总成人PAP1（REG3α）的工具包</td><td> MBL国际</td><td> 5323 </td><td></td></tr><tr><td> AB比赛UNIVERSAL套件</td><td> MBL国际</td><td> 5310 </td><td></td></tr><tr><td>人类sTNFRI/TNFRSF1A DUOSET </td><td> R＆D Systems公司</td><td> DY225 </td><td></td></tr><tr><td>人类Trappin-2/Elafin DUOSET </td><td> R＆D施茎</td><td> DY1747 </td><td></td></tr><tr><td> 96 - 孔聚苯乙烯锥形底板</td><td> Thermo Scientific的</td><td> 249570 </td><td>用于等离子体源板</td></tr><tr><td> Costar公司半井的高结合96 - 孔板</td><td>康宁</td><td> 3690 </td><td>对于IL-2Rα，HGF，TNFR1和elafin ELISA试剂盒</td></tr><tr><td> Nunc公司384给MaxiSorp板</td><td> Nunc公司</td><td> 464718 </td><td>对于REG3αELISA </td></tr><tr><td>胎牛血清磷酸盐缓冲液，1个</td><td> Thermo Scientific的</td><td> SH30256.02 </td><td></td></tr><tr><td>牛血清白蛋白组分V，热休克治疗</td><td> Fisher Scientific则</td><td> BP1600-100 </td><td></td></tr><tr><td>在TBS的拦截器BLOTTO </td><td> Thermo Scientific的</td><td> 37530 </td><td> IL-2Rα肝细胞生长因子和TNFR1酶联免疫吸附阻断剂</td></tr><tr><td> Dulbecco的磷酸盐缓冲液</td><td> Gibco公司</td><td> 21600-069 </td><td>洗涤缓冲液，肝细胞生长因子IL-2Rα，Elafin和TNFR1酶联免疫吸附</td></tr><tr><td> TMB过氧化物Susbtrate </td><td> Kirkegaard和佩里实验室</td><td> 50-76-00 </td><td></td></tr><tr><td>吐温20 </td><td> Acros Organics公司</td><td> 233362500 </td><td>洗涤缓冲液，肝细胞生长因子IL-2Rα，Elafin和TNFR1酶联免疫吸附</td></tr><tr><td>硫酸</td><td> Sigma-Aldrich公司</td><td> 84720 </td><td> （稀释到2N）的一站式解决方案</td></tr></tbody></table>

high throughput sequential elisa for validation of biomarkers of acute graft-versus-host disease

偏发现蛋白质组学研究策略有可能确定大量的新型生物标志物的检测，在临床上可以提高诊断和预后，并可能有助于指导治疗干预。当大量的候选蛋白，它可能是难以验证的候选生物标志物的及时和有效的方式，从患者的血浆样本是事件驱动的，有限的，不可替代的量，如在发病的急性移植物抗宿主病（GVHD），异基因造血干细胞移植（HSCT）的一个潜在的危及生命的并发症。 在这里，我们描述的过程中，执行6验证的移植物抗宿主病（GVHD）的蛋白质的市售的ELISA试剂盒：IL-2Rα5星 ，TNFR1 6，肝细胞生长因子7，IL-8 8，elafin 2，和REG3α3（也称为PAP1）以顺序的方式，以尽量减少冻融循环，解冻的血浆和血浆的使用。对于此过程中，我们进行了酶联免疫吸附顺序确定样品稀释倍数作为建立在我们的实验室制造商ELISA试剂盒和协议细微的调整，以方便最优顺序ELISA性能。由此产生的血浆生物标志物的浓度可以被编译和重大的发现在患者队列分析。虽然这些生物标志物是目前仅用于研究目的，将其纳入临床护理，目前正在临床试验中。 此技术也可应用于在同一样品上执行多个感兴趣的蛋白/细胞因子ELISA试剂盒（）提供的样品不需要与其它试剂混合。如果ELISA试剂盒不配备预涂覆板，96孔半孔板或384孔板可以使用，以进一步减少使用的样本/试剂。

<html>生物标志物的工作流程和时间安排详列于表1和表2分别。一旦完成后，6个不同的蛋白质的浓度，现在已经被量化相同的血浆样品中，总共使用了150μL血浆。电镀样品一式两份，每份每次测试允许内部的质量保证，与CV的小于10％是最佳的。如果执行的顺序ELISA在多个板，一致的高标准的光密度是优选的，并允许改进的板间的测量的可靠性;看评估在ELISA性能不一致的（ 图2），板之间的可比较的标准曲线外径。在我们的实验室中观察到的各生物标记物的开发时间，使用四甲基联苯胺比色法基板和高浓 ​​度的外径在表3中列出。 tp_upload/4247/4247fig1.jpg“ALT =”图1“/&gt; 图1 IL-2Rα，REG3α和肝细胞生长因子ELISA试剂盒的工作流程。血浆样品后已镀上的IL-2Rα的酶联免疫吸附试验板，它被回收，使稀释的源其他的ELISA板。对于肝细胞生长因子酶联免疫吸附，血浆回收1:6稀释平板IL-8的准备。 <a href="http://www.jove.com/files/ftp_upload/4247/4247fig1large.jpg" target="_blank">点击查看大图</a> 。 <img src="/files/ftp_upload/4247/4247fig2.jpg" alt="图2"/> 图2的7种不同的ELISA板，测量对应于测试的初始REG3αGI移植物抗宿主病的生物标志物报告3 1084例REG3α浓度的标准曲线的光学密度。板块之间的一致消耗臭氧层物质保证板块之间的一致的蛋白浓度测量。蛋白质的浓度血浆样品中通过对比样品的光密度的标准曲线的光密度计算。 <table border="1"><tbody><tr><td> 实验第0天 </td><td> 1。准备样品</td></tr><tr><td></td><td> 2。 IL-2Rα，HGF和REG3α的捕获抗体</td></tr><tr><td> 实验第1天 </td><td> 1。 IL-2RαELISA的</td></tr><tr><td></td><td> 2。 REG3αELISA </td></tr><tr><td></td><td> 3。 HGF ELISA（通过抽样电镀） </td></tr><tr><td></td><td> 4。 Elafin捕获抗体和肿瘤坏死因子受体</td></tr><tr><td> 实验第2天 </td><td> 1。 HGF ELISA完成</td></tr><tr><td></td><td> 2。 Elafin ELISA </td></tr><tr><td></td><td> 3。 TNFR1 ELISA </td></tr><tr><td></td><td> 4。 IL-8 ELISA </td></tr><tr><td></td><td> 5。重新冻结未使用的血浆</td></tr></tbody></table> 表1。移植物抗宿主病生物标记物的工作流程概观<table border="1"><tbody><tr><td colspan="2"> 0天 </td><td colspan="3"> 样品制备和隔夜捕捉身体孵化 </td><td></td><td></td><td></td></tr><tr><td colspan="2"> ELISA </td><td> IL-2Rα </td><td> REG3α </td><td> HGF </td><td> Elafin </td><td> TNFR1 </td><td> IL-8的 </td></tr><tr><td> 时间（小时） </td><td> 0.0 </td><td>闭塞</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 1.0 </td><td>样品镀</td><td></td><td></td><td></td><td></td> &lt;TD&gt; </td></tr><tr><td></td><td> 3.0 </td><td>回收血浆样品，检测抗体</td><td> ;样品制备（1:10稀释） </td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 4.0 </td><td></td><td>样品镀（1:10） </td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 5 </td><td>链霉亲和素-HRP </td><td>检测抗体</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 5.5 </td><td> TMB </td><td>链霉亲和素-HRP </td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 6 </td><td>读板机</td><td> TMB </td><td>封锁，准备样品（1:2稀释） </td><td></td><td></td><td></td></tr><tr><td></td><td> 6.5 </td><td></td><td>读板机</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 7 </td><td></td><td></td><td>样片镀（1:2） </td><td>捕获抗体（过夜培养） </td><td>捕获抗体（过夜培养） </td><td></td></tr><tr><td> 第2天 </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 时间（小时） </td><td> 0.0 </td><td></td><td></td><td>回收血浆，检测抗体</td><td>封锁，准备样品（1:20） </td><td></td><td></td></tr><tr><td></td><td> 1.0 </td><td></td><td></td><td></td><td>样品电镀（1:2） </td><td> ; 1:20的样品进一步稀释到1:25稀释</td><td></td></tr><tr><td></td><td> 2.0 </td><td></td><td></td><td> HRP </td><td></td><td>样品电镀（1:25） </td><td></td></tr><tr><td></td><td> 2.5 </td><td></td><td></td><td> TMB </td><td></td><td></td><td></td></tr><tr><td></td><td> 3.0 </td><td></td><td></td><td>读板机</td><td>检测抗体</td><td></td><td></td></tr><tr><td></td><td> 3.5 </td><td></td><td></td><td></td><td></td><td></td><td>准备样品（1:6稀释） </td></tr><tr><td></td><td> 4.0 </td><td></td><td></td><td></td><td></td><td>检测抗体</td><td>样品电镀（1:6） </td></tr><tr><td></td><td> 5 </td><td></td><td></td><td></td><td> HRP </td><td></td><td></td></tr><tr><td></td><td> 5.5 </td><td></td><td></td><td></td><td> TMB </td><td></td><td></td></tr><tr><td></td><td> 6 </td><td></td><td></td><td></td><td>读板机</td&gt; <td> HRP </td><td>检测抗体</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td> TMB </td><td></td></tr><tr><td></td><td> 7 </td><td></td><td></td><td></td><td></td><td>读板机</td><td> TMB </td></tr><tr><td></td><td> 7.5 </td><td></td><td></td><td></td><td></td><td></td><td>读板机</td></tr><tr><td colspan="2"> 完成后 </td><td colspan="6">原料血浆替换装冻结以备后用</td></tr></tbody></table> 表2。进行酶联免疫吸附的时间表。 <table border="1"><tbody><tr><td></td><td> 等离子稀释因子 </td><td> 高标准浓度 </td><td> 基板的开发时间（分钟） </td><td> 高OD </td><td> 曲线 </td></tr><tr><td> IL-2Rα </td><td> 1:1 </td><td> 2000皮克/毫升</td><td> 5 </td><td> 1 </td><td>线性</td></tr><tr><td> HGF </td><td> 1:2 </td><td> 4000皮克/毫升</td><td> 22 </td><td> 2.1 </td><td> 4 - 参数</td></tr><tr><td> IL-8的</td><td> 1:6 </td><td> 200皮克/毫升</td><td> 12 </td><td> 2.7 </td><td> 4 - 参数</td></tr><tr><td> REG3α </td><td> 1:10 </td><td> 100纳克/毫升</td><td> 12 </td><td> 1.7 </td><td> 4 - 参数</td></tr><tr><td> Elafin </td><td> 1:20 </td><td> 2000皮克/毫升</td><td> 20 </td><td> 1.9 </td><td> 4 - 参数</td></tr><tr><td> TNFR1 </td><td> 1:25 </td><td> 800皮克/毫升</td><td> 8 </td><td> 2.7 </td><td>线性</td></tr></tbody></table> 表3。ELISA详情6移植物抗宿主病（GVHD）的生物标志物。

Watch this Scientific Journal Video about High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease at JoVE.com

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

多个候选生物标志物的高吞吐量的验证可以通过顺序ELISA进行，以尽量减少冷冻/解冻循环和使用珍贵的血浆样品。在这里，我们演示了如何依次进行验证六种不同的血浆生物标志物的酶联免疫吸附<sup&gt; 1-3</sup的移植物抗宿主病（GVHD）<sup&gt;</sup上相同的血浆样品。

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 ...

high-throughput-sequential-elisa-for-validation-biomarkers-acute

Research

JoVE Journal

Medicine

15.7K 次观看.  University of Michigan. 多个候选生物标志物的高吞吐量的验证可以通过顺序ELISA进行，以尽量减少冷冻/解冻循环和使用珍贵的血浆样品。在这里，我们演示了如何依次进行验证六种不同的血浆生物标志物的酶联免疫吸附 1-3

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

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

一种高通量自动化的平台，制造蛋白质治疗的细胞系发展

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

科研

医学

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

老鼠的眼睛剜除术的远程高通量的表型

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

在诱导小鼠心肌梗死的微创手术过程

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

发现疾病相关的α突触核蛋白通过增强ELISA在转基因小鼠表达人A53T的突变脑α突触核蛋白

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

高通量，复用和有针对性的蛋白质组学分析脑脊液量化神经退行性生物标志物和载脂蛋白E亚型状态

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 对高脂饮食喂养小鼠体内葡萄糖代谢的影响研究 (英文)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...