NanoManufacturing Innovation Consortium for funding support.

1.准备解决方案和蛋白质样品的SPRI的<ol><li>制备100毫升含有10mM磷酸盐，150mM氯化钠，pH为7.4低盐磷酸盐缓冲盐水（PBS）溶液。 </li><li>制备50ml含有10mM磷酸盐，750mM的氯化钠，pH为7.4的高盐PBS溶液。 </li><li>准备5毫升的10mM醋酸钠。 </li><li>制备的5mg / ml的牛血清白蛋白（BSA）稀释在低盐PBS溶液股票。 </li><li>以1微克/毫升低盐PBS溶液的浓度制备的重组人生长激素（rhGH治疗）的储备溶液。 </li><li>制备储备溶液的抗重组人生长激素和阴性对照抗体以100μg/ ml的浓度。 </li></ol> 2.准备SPRI芯片抗体芯片<ol><li>清洁的120ml的稳定化的食人鱼溶液通过超声处理芯片（3:1浓硫酸至30％过氧化氢）90分钟，在50℃，并按照通过漂洗和超声处理与水5分钟。然后，冲洗芯片用乙醇和干燥与氮气流。 </li><li>将金芯片中的UV /臭氧室中30分钟，以除去任何污染物。 </li><li>加入150毫克的11-巯基十一酸到20ml乙醇中，在试管中含有搅拌棒和管帽。 </li><li>放置芯片在试管和微波管/芯片在50瓦和50℃进行5分钟。 </li><li>片冲洗用乙醇浸泡在乙醇5分钟。 </li><li>用水冲洗遵循并在水中浸泡5分钟。 </li><li>加入150毫克1-乙基-3-（3-二甲基氨基丙基）碳二亚胺到20ml水在试管中包含搅拌棒和管帽。微波芯片如在步骤2.4。 </li><li>片冲洗水和在水中浸泡5分钟。 </li><li>添加150毫克N-羟基琥珀酰亚胺在20毫升水在试管中包含搅拌棒和管帽。微波芯片如在步骤2.4。 </li><li>片冲洗水和在水中浸泡5分钟。 </li><li>编写，J重新达到250μM聚乙二醇（PEG，800道尔顿）的20ml水在试管中包含搅拌棒和管帽。微波芯片如在步骤2.4。 注意:微波PEG800所述传感器的表面上被执行作为封闭步骤以防止非特异性血清蛋白和量子点复合物的结合实验中以后使用。这是通过PEG800反应与非占用裸金点在芯片上实现。 </li><li>片冲洗水和在水中浸泡5分钟。 </li><li>制备的抗生长激素抗体溶液和抗免疫球蛋白G（抗-IgG）阴性对照液15微克/毫升，并添加10微升每种抗体溶液到一个井在384孔板中。 </li><li>设计在微阵列（4×7象限每个样品）和现货芯片的斑点图案，使用500微米的特氟隆抗体放倒针。 </li><li>孵育斑点芯片为2小时，在室温，75％或更大的潮湿的气氛下。 </li><li>冲洗，浸泡在水中芯片5分钟。然后，干燥的芯片与氮气流。 </li></ol> 3. SPRI实验设置和阻塞<ol><li>初始化工具，然后选择目录。选择实时照相机和启动注射泵以1ml /分钟进行10毫升脱气水中。将芯片插入仪器，并保持注水，直到所有的气泡被删除。 </li><li>漂洗芯片，10毫升水后，启动图像采集，并按照通过切换缓冲器低盐PBS中，并允许它以1毫升5 ml和然后缓慢流速运行/分钟降至20μl/ min的20分钟。 </li><li>选择高对比度图像，其示出有斑点到芯片上的固定化抗体。 </li><li>选择和定义对应于所述固定化抗体的400微米的个体圆形斑点。 </li><li>经过仪器跟踪等离子体曲线，选择一种工作的角度，有在反射率曲线的最大斜率。 </li><li>低S的流动的运行缓冲液在20μL/分钟按alt PBS通过该系统直到所有选定点的信号稳定。 </li><li>加载的BSA（100微克/毫升）的运行缓冲液进样品环（150微升）与在装载位置的喷射阀。然后切换阀到注入位置到该溶液到流动池注入。 注意:BSA被注入到共价结合到胺反应性表面，剩余的未结合的BSA将洗掉表面通过缓冲冲洗。 </li><li>注入150微升10mM醋酸钠在表面上的溶液，并按照由150微升注射高盐PBS中。 </li><li>注入150微升乙醇胺（1毫米）的停用胺活性表面。 </li><li>注入150微升10mM醋酸钠洗表面。 </li><li>然后，切换运行缓冲到PBS中50μg/ ml的BSA以250微升/分钟，共5毫升的流速。 注:50微克/毫升牛血清白蛋白加入到运行缓冲液为的河畔附加阻挡面由非共价地占据非特异性位点，这是为了进一步减少非特异性血清蛋白结合到表面。 </li><li>变化流速5微升/分钟，并允许其于20分钟稳定。 </li></ol> 4. SPRI检测人体生长激素<ol><li>制备重组人生长激素的设定浓度的溶液（30,000微克/毫升; 250微克/毫升; 25皮克/毫升; 2.5微克/毫升和0.25微克/毫升）的10％血清和在PBS中稀释含有1mg / ml BSA中。 </li><li>制备2:通过加入1微升生物素1溶液标记的抗重组人生长激素的检测抗体（6μM）至3微升链霉的在0.5 ml离心管涂覆近红外线的量子点（1μM）和温育30分钟进行有效耦合。 </li><li>注入150微升hGH的飙升注射环人血清的解决方案。这导致随后缓慢滴从非特异性结合的血清漂洗掉表面在信号的强劲增长。 </li><li>一旦签收人稳定，用450毫摩的氯化钠加入到运行缓冲溶液清洗的表面上。 </li><li>稀释的抗重组人生长激素的检测抗体和量子点的溶液来为10nM（2:1）的浓度与运行缓冲液（PBS中50μg/ ml的BSA）和注入流动池。 </li></ol> 5. SPRI数据分析注意:在注射的重组人生长激素的掺入人血清和量子点增强剂，增加的反射率变化的发生是由于在等离子体曲线的扩增移位。这导致了差的图象，显示的灰度的图像进行关联的芯片上的信号。 <ol><li>将数据导入到一个数据分析程序。绘制SPRI信号（反射率％）与时间（s），并确定该高盐洗涤后的抗重组人生长激素和阴性对照抗体斑点之间的差异。 </li></ol> 6. ELISA协议（第1天） <ol><li>存储所有检测组件包括我n中的重组人生长激素ELISA试剂盒在2-8℃。这包括抗生长激素抗体包被的孔板，标准（0-5）在羊血清，对照（1和2）在人血清，共轭缓冲液，（200倍）洗涤缓冲液，显色剂TMB（四甲基联苯胺）和停止试剂。 </li><li>设置的试剂，并按照在商业ELISA试剂盒协议描述的方法。 </li><li>第一，允许防生长激素抗体包被的96孔温热至RT打开箔包之前。 </li><li>然后取出的8孔条带所需的数字用于分析和重新密封含有剩余孔中，并存储在2-8℃下的箔包。 </li><li>由重建在2ml蒸馏水标准＃0制备的标准，在1ml蒸馏水标准（1-5），并在1毫升蒸馏水的对照组（1和2）。下面是一个标准和控制（表1）中相应的浓度的一个表: </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-与-next.within页="总是"&gt; <tbody><tr><td> 标准 </td><td> 浓度（ng / ml）的 </td></tr><tr><td> 0 </td><td> 0 </td></tr><tr><td> 1 </td><td> 0.17 </td></tr><tr><td> 2 </td><td> 0.94 </td></tr><tr><td> 3 </td><td> 2.63 </td></tr><tr><td> 4 </td><td> 10.4 </td></tr><tr><td>五</td><td> 26.9 </td></tr><tr><td>控制＃1 </td><td> 1.22±0.33 ng / ml的</td></tr><tr><td>控制＃2 </td><td> 4.97±1.25 ng / ml的</td></tr></tbody></table> 表1浓度的标准和控制包括在商业ELISA试剂盒。 <ol start="6"><li>准备其由所述重组人生长激素的激素，在10％的人血清以下列浓度（表2）的样品: </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> &lt;STRONG&gt;样品</td><td> 浓度（ng / ml）的 </td></tr><tr><td> 1 </td><td> 0.025 </td></tr><tr><td> 2 </td><td> 0.2 </td></tr><tr><td> 3 </td><td> 0.5 </td></tr><tr><td> 4 </td><td> 2.5 </td></tr><tr><td>五</td><td> 10 </td></tr><tr><td> 6 </td><td>三十</td></tr></tbody></table> 表2浓度在10％的血清中制备的重组人生长激素的样品。 <ol start="7"><li>添加50微升每个标准，对照和样品，每孔（一式三份）。密封的水井，并在轻轻摇动的标准，控制和重组人生长激素样品O / N孵育4℃。 </li></ol> 7. ELISA协议（2日） <ol><li>从摇床中取出板，并允许它温热至室温（15-20分钟）之前，继续进行测定。 </li><li>卸下防重组人生长激素，HRP，共轭缓冲液，洗涤液（200倍），显色TMB（四甲基联苯胺），一从冰箱D停止试剂，并允许温热至室温。 </li><li>试剂的准备:（根据制造商的规格） <ol><li>稀释反的rhGH-HRP 40X与共轭缓冲液中。通过用蒸馏水稀释200倍制备洗涤缓冲液。 </li></ol></li><li>加入50微升的抗生长激素-HRP结合到每口井，封闭的井和在室温下孵育30分钟，同时轻轻摇晃。 </li><li>滗从孔中的溶液并反转板轻轻吸干到吸收组织。然后加入200微升洗涤缓冲液到每个孔中。 </li><li>倒出洗涤液与自来水干到吸收组织。重复此步骤3次。 </li><li>加入100μl显色到每口井在15分钟后的清洗步骤。孵育在室温下30分钟，在黑暗中，而轻轻颤抖。该解决方案将从无色变为蓝色。 </li><li>加入100微升终止试剂在每孔中。这些解决方案从蓝色变为黄色。随即，阅读日Ë吸光度用酶标仪各孔在450nm处。 </li><li>绘制标准曲线所提供的标准（0-5）。然后绘制的rhGH的光密度，在10％的血清样品相对于各样品的浓度。 </li></ol>

<ol>
	<li>Popii, V., Baumann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+measurement+of+growth+hormone.">Laboratory measurement of growth hormone.</a> Clinica Chimica Acta. 350 (1-2), 1-16 (2004).</li><li>Higham, C. E., Trainer, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+hormone+excess+and+the+development+of+growth+hormone+receptor+antagonists.">Growth hormone excess and the development of growth hormone receptor antagonists.</a> Exp Physiol. 93 (11), 1157-1169 (2008).</li><li>S&#246;nksen, P., Salomon, F., Cuneo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+effects+of+hypopituitarism+and+acromegaly.">Metabolic effects of hypopituitarism and acromegaly.</a> Horm Res. 36, 27-31 (1991).</li><li>Molitch, 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=Evaluation+and+Treatment+of+Adult+Growth+Hormone+Deficiency:+An+Endocrine+Society+Clinical+Practice+Guideline.">Evaluation and Treatment of Adult Growth Hormone Deficiency: An Endocrine Society Clinical Practice Guideline.</a> J Clin Endocrinol Metab. 91 (5), 1621-1634 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The World Anti-Doping Code: The 2015 Prohibited List International Standard. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The 2014 prohibited list world anti-doping code. , (2014).</li><li>Langkamp, M., Weber, K., Ranke, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+growth+hormone+measurement+by+means+of+a+sensitive+ELISA+of+whole+blood+spots+on+filter+paper.">Human growth hormone measurement by means of a sensitive ELISA of whole blood spots on filter paper.</a> Growth Horm IGF Res. 18 (6), 526-532 (2008).</li><li>Rich, R. L., Myszka, 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=Survey+of+the+year+2007+commercial+optical+biosensor+literature.">Survey of the year 2007 commercial optical biosensor literature.</a> J Mol Recognit. 21 (6), 355-400 (2008).</li><li>Homola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+of+surface+plasmon+resonance+biosensors.">Present and future of surface plasmon resonance biosensors.</a> Anal Bioanal Chem. 377 (3), 528-539 (2003).</li><li>Hoa, X. D., Kirk, A. G., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Integrated+Surface+Plasmon+Resonance+Biosensors:+A+Review+of+Recent+Progress.">Toward Integrated Surface Plasmon Resonance Biosensors: A Review of Recent Progress.</a> Biosens Bioelectron. 23 (2), 151-160 (2007).</li><li>de Juan-Franco, E., Rodr&#236;guez-Frade, J. M., Mellado, M., Lechuga, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+a+SPR+immunosensor+for+the+simultaneous+detection+of+the+22K+and+20K+hGH+isoforms+in+human+serum+samples.">Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples.</a> Talanta. 114, 268-275 (2013).</li><li>de Juan-Franco, E., Caruz, A., Pedrajas, J. R., Lechuga, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+antibody+immobilization+using+a+protein+A-gold+binding+domain+fusion+protein+for+enhanced+SPR+immunosensing.">Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.</a> Analyst. 138 (7), 2023-2031 (2013).</li><li>Du, H., 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=Immunological+screening+and+characterization+of+highly+specific+monoclonal+antibodies+against+20+kDa+hGH.">Immunological screening and characterization of highly specific monoclonal antibodies against 20 kDa hGH.</a> Bioanalysis. 4 (17), 2161-2168 (2012).</li><li>Trevi&#241;o, J., Calle, A., Rodr&#236;guez-Frade, J. M., Mellado, M., Lechuga, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance+immunoassay+analysis+of+pituitary+hormones+in+urine+and+serum+samples.">Surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples.</a> Clin Chim Acta. 403 (1-2), 56-62 (2009).</li><li>Wu, Z., Bidlingmaier, M., Dall, R., Strasburger, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+doping+with+human+growth+hormone.">Detection of doping with human growth hormone.</a> Lancet. 353 (9156), 895 (1999).</li><li>Malic, L., Sandros, M. G., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designed+biointerface+using+near-infrared+quantum+dots+for+ultrasensitive+surface+plasmon+resonance+imaging+biosensors.">Designed biointerface using near-infrared quantum dots for ultrasensitive surface plasmon resonance imaging biosensors.</a> Anal. Chem. 83 (13), 5222-5229 (2011).</li><li>Vance, S. A., Sandros, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zeptomole+Detection+of+C-Reactive+Protein+in+Serum+by+a+Nanoparticle+Amplified+Surface+Plasmon+Resonance+Imaging+Aptasensor.">Zeptomole Detection of C-Reactive Protein in Serum by a Nanoparticle Amplified Surface Plasmon Resonance Imaging Aptasensor.</a> Sci. Rep. 4 (5129), 1-7 (2014).</li><li>Uludag, Y., Tothill, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+biomarker+detection+in+serum+samples+using+surface+plasmon+resonance+and+quartz+crystal+microbalance+sensors+with+nanoparticle+signal+amplification.">Cancer biomarker detection in serum samples using surface plasmon resonance and quartz crystal microbalance sensors with nanoparticle signal amplification.</a> Anal. Chem. 84 (14), 5898-5904 (2012).</li><li>&#352;pringer, T., Homola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofunctionalized+gold+nanoparticles+for+SPR-biosensor-based+detection+of+CEA+in+blood+plasma.">Biofunctionalized gold nanoparticles for SPR-biosensor-based detection of CEA in blood plasma.</a> Anal Bioanal Chem. 404 (10), 2869-2875 (2012).</li></ol>

M.G. Sandros is a consultant for Horiba Scientific. She has received honoraria for participation in oral presentations from: Horiba Scientific and Syngenta. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript

生长激素，一种自然产生的激素水平不规则，已与影响人体生长发育的许多内科疾病。另外，生长激素的外源性给药通常所使用的运动员，即使它是被禁止的，作为掺杂剂，以提高它们的性能。在从内源性的形式区分外源性生长激素的难度检测生长激素滥用的结果挑战。这样，目前批准的技术，用于检测外源性的hGH依赖于测量的22kDa hGH的同种型的比率相对于20kDa的同种型。由于多个hGH的测量亚型测试需求同时亚型时间在广泛的浓度在短期内，因此，我们认为SPRI平台绝配。此外，内源性的hGH水平波动，以在血流中一个非常低的水平（0.03毫微克/毫升），因此，检测系统必须能够舒适地测量该范围内的高菌ecificity。其结果是，我们还研究了在本研究纳米SPRI的潜力作为hGH的诊断工具，并直接与SPRI和经典免疫测定的ELISA进行了比较。 基于从本研究获得的结果，在SPRI和纳米SPRI方法的主要优点是，重组人生长激素的浓度可以以更快的方式相比，更常规的ELISA方法进行测定。一个标准的持续时间，用于测量的rhGH水平在一个样品与直接检测方法为1小时，而纳米SPRI需要2小时，由于在这个过程中的其他步骤。总体而言，与SPRI和纳米单层屋面实验中，注射样品之前，校准步骤，强烈推荐。此外，注射像人血清的结果在一些非特异性相互作用，结果就必须注入高盐洗涤缓冲液，以仅露出的特异性相互作用的粗样品。还值得一提的是，洗涤步骤是绝对必要的引入阻断分子与传感器表面之后，以去除未结合的分子。如用于ELISA时间要求大得多（〜16-18小时）为一个样品的分析。需要较长的温育时间作为测定的灵敏度被增强特别适用于本研究中，作为焦点是比较检测的下限。 表面化学的选择将变化从一个应用到另一个，这可以被实现为在SPRI技术的限制之一。在这项研究中，大范围和化学接头和阻断分子的组合进行了评估，实现了正确的组合，以便观察的rhGH的最佳结合效率到传感器表面。例如，在这项研究中，BSA和PEG的组合很好服务在尽量减少非特异性相互作用。然而，在先前的研究中17，其中捕获配位体是一种适体，PEG-单独担任最好阻挡分子。变量分析物的配体影响结合效率也取决于pH值，缓冲剂和温度。因此，与任何应用程序，这些变量需要优化。此外，重要的是，以确定配体与芯片表面的最佳点样浓度。滴定实验与一系列固定的配体的浓度正在开始研究之前进行的。至于ELISA，在该过程中至关重要的一步是通过点击微孔板倒出从水井的洗涤液，因为这确保无残留的液体是吃剩的。除去缓冲液洗涤用吸移管是不够的，因为任何残留液体干扰了靶样品的信号读取。 在参考灵敏度，酶联免疫吸附（1纳克/毫升）相媲美SPRI（3.61毫微克/毫升），但纳米SPRI（9.20微克/毫升）通过三个数量级提高了灵敏度，由此使得测量在重组人生长激素的下部生物水平0.03纳克/毫升。正如我们先前报道的16,17，由NanoEnhancers施加的信号增强归因于质量负荷效果和强耦合存在的NIR荧光团和传播表面等离子体之间的金膜的纳米结构。即使，纳米SPRI增加了一个额外的步骤的程序，该敏感度可以加宽SPRI技术在各种网点的应用程序。 SPRI提供抗体/重组人生长激素相互作用的科学家们一个完整的动力学特征（K D，K 一和K D），而ELISA可以仅报告亲和度值。变化（CV）的系数为下面SPRI（4.1％）和ELISA（6.5％）10％，这表明良好的再现性。在ELISA和SPRI亲和力值是不同的，因为固定在传感器芯片上的捕获抗体是来自固定在ELISA 96孔平板的抗体不同。作为用于纳米SPRI更高的CV值（20％）进行了观察。有可作为埃罗源贡献几个参数RS为CV的决心。例如，随着纳米SPRI实验分析物的浓度低得多正在测量，加入NanoEnhancers的增加在该过程的另一个步骤，并手动进行实验。纳米单层屋面和ELISA之间很好的相关性为重组人生长激素的尖刺人血清的检测达到了。最后，酶联免疫吸附可以是一个可靠的技术然而该方法本身是耗时的，这使得它在要求实时监测和复用的情况下很难使用，因为它与hGH的情况。此外，在该研究，SPRI提供超过ELISA，这不是直接调查了更吸引人的特点，是测量实时同时数百相互作用的能力。因此，在未来，纳米SPRI方法将被评估，以检测存在于血清中的各种浓度的多种生物标志物同时在实时（复用），以考察其作为一种可行的临床诊断工具的潜能。

人体生长激素（hGH）是由垂体生成和直接释放到血液中一个191个氨基酸的肽（22 kDa）的。下丘脑肽生长激素释放激素（GHRH）和生长激素之间的相互作用诱导hGH的搏动分泌物。其结果是，hGH的水平从高点在50-100 ng / ml的在0.03毫微克/毫升范围1低点而变化。缺乏或过量的hGH在体内会引起强烈的各种异常的生理症状。例如，hGH的过量水平可导致巨人症和2型糖尿病3。 hGH的耗尽水平引起低血糖的新生儿，而弱骨密度和抑郁症的成年人4。 同时减少体内脂肪生长激素（重组人生长激素）的重组形式的管理提高肌肉质量。因此，这种物质成为了专业和业余运动员的首选药物，因为它提高赋予副词体力antage竞技体育。重组人生长激素被禁止由世界反兴奋剂机构（WADA）5,6和很大的努力受到国际学者一直专注于开发测试，可以检测到它的存在或合成代谢作用。 酶联免疫吸附测定（ELISA）已为hGH的全血7测定的首选方法。虽然，酶联免疫吸附是一种可靠的技术，提供良好的灵敏度和选择性，它是相对时间和劳动密集的。另外，ELISA采用酶标记依赖于生长激素的间接检测。与此相反，表面等离子体共振（SPR）允许检测hGH的情况下直接实时使用标签。后面的SPR检测原理涉及的感测表面包括涂覆有薄金属层（金或银）的棱镜的;当单色偏振光与金属表面相互作用，"表面等离子体"产生。分析物的结合与固定在该金属表面上的表面受体扰乱从而产生偏移谐振浸，然后可将其相关的被分析物浓度的谐振条件。基于SPR生物传感器现在市售，提供实时，无标记技术来监测生物分子结合事件和生化反应8-10。最近，SPRI响应于需要多路复用被开发（即，监测同时对多个结合事件），这是不可能的古典的SPR生物传感器。因此，SPRI已成为一种工具，同时监视多个结合事件。当前SPRI系统是基于其在一个特定的角度和波长10激发的光的表面的显微成像。该图像然后被捕获到电荷耦合器件（CCD）阵列。 迄今为止，已经开发了用于检测的hGH 11-14少数基于SPR测定。一个特别的策略被称为同种型的方法15，依赖于22 kDa的hGH的总的hGH的比例的检测中，如后外源性生长激素给药非22-kDa的内源性水平下降。最近，德涓-法等 11报道了一个基于SPR的免疫传感器的发展，为选择性检测的22 kDa的和20kDa hGH的同种型的人类血清样品中的。具体到每个同种型的单克隆抗体直接固定在金传感器同时允许两者的异构体的测量在单个注射检测限在0.9纳克毫升-1。可替代地，SPR已经用于筛选抗体以高特异性与hGH 13。如果目标分析物的浓度低于检测SPRI系统的限制（&lt;纳米），人们必须诉诸通过纳米颗粒的利用率（纳米SPRI）扩增SPRI信号。这种基于SPR的扩增已经很好记载在文献16-19中Various类型的分析物和表面。 在这项工作中，SPRI和纳米SPRI基于生物传感器的分析潜力进行了检查，特别是对重组人生长激素中加标人血清的检测，和其检测直接能力的ELISA比较。下面的参数进行审查，认为:检测时间，灵敏度，动力学曲线，重现性和特异性。

<table><tbody><tr><td>Somatotropin Growth Hormone (GH1) capture antibody&amp;nbsp;</td><td>Acris Antibodies</td><td>DM1015</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Fisher Bioreagents&amp;nbsp;</td><td>BP671-10</td><td></td></tr><tr><td>Biotin labeled Somatotropin Growth Hormone (GH1) detection antibody&amp;nbsp;</td><td>Acris Antibodies</td><td>AM09304BT-N</td><td></td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide&amp;nbsp;</td><td>TCI America</td><td>D1601</td><td></td></tr><tr><td>Ethanolamine</td><td>Acros Organics&amp;nbsp;</td><td>141-43-5</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Chemicals&amp;nbsp;&amp;nbsp;</td><td>BP2818-4</td><td></td></tr><tr><td>Human HGH ELISA Kit&amp;nbsp;</td><td>invitrogen&amp;nbsp;</td><td>KAQ1081</td><td></td></tr><tr><td>Human Serum&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>H4522&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Rabbit IgG Antibody&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>I5006</td><td></td></tr><tr><td>11-mercaptoundecanoic acid&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>450561</td><td></td></tr><tr><td>Nanostrip&amp;nbsp;</td><td>Cyantek&amp;nbsp;</td><td></td><td></td></tr><tr><td>N-hydroxysuccinimide&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>130672</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Invitrogen&amp;nbsp;</td><td>00-3002</td><td></td></tr><tr><td>Polyethylene glycol (800 Da)&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>729108</td><td></td></tr><tr><td>Recombinant Human Growth Hormone&amp;nbsp;</td><td>Calbiochem</td><td>869008</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma Aldrich&amp;nbsp;</td><td>127-09-3</td><td></td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Fisher Chemicals&amp;nbsp;&amp;nbsp;</td><td>7647-14-5</td><td></td></tr><tr><td>Streptavidin coated near infrared quantum dots&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>Q10171MP</td><td></td></tr><tr><td>UV/Ozone Procleaner</td><td>Bioforce Nanosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>Microwave reactor&amp;nbsp;&amp;nbsp;</td><td>CEM Corporation</td><td></td><td>Discover system&amp;nbsp;</td></tr><tr><td>SPRi-Arrayer&amp;nbsp;&amp;nbsp;</td><td>LabNext Xactll Microarray System&amp;nbsp;</td><td></td><td></td></tr><tr><td>SPR biochip</td><td>HORIBA&amp;nbsp;</td><td></td><td></td></tr><tr><td>SPRi-Lab+</td><td>HORIBA</td><td></td><td></td></tr><tr><td>Synergy Mx Multimode Microplate reader&amp;nbsp;</td><td>BioTek&amp;nbsp;</td><td></td><td></td></tr><tr><td>ScrubberGen</td><td>HORIBA</td><td></td><td>Data Analysis software&amp;nbsp;</td></tr></tbody></table>

comparative analysis of human growth hormone in serum using spri, nano-spri and elisa assays

灵敏的和选择性的方法，人类生长激素（hGH）的检测在宽范围的浓度（高含量的50-100纳克毫升- 1和 0.03纳克毫升最低水平- 1）在循环血液是必要的，因为可变的水平可表明改变生理。例如，在儿童期出现的生长障碍，可以通过测量血液中的hGH水平进行诊断。此外，重组生长激素在体育滥用不仅提出了一个道德问题，也呈现给施虐者严重的健康威胁。用于测量的hGH滥用一个流行的策略，依赖于22 kDa的hGH的总的hGH的比例的检测中，如后外源重组的hGH（重组人生长激素）给药非22 kDa的内源性水平下降。 表面等离子体共振成像（单层屋面）是一种分析工具，可以直接（无标记）的监测和生物分子相互作用的可视化通过记录折射IND的变化前相邻实时传感器表面。与此相反，使用频率最高的比色法，酶联免疫吸附测定（ELISA）使用酶标记的检测抗体添加底物诱导的颜色变化的后来间接测量分析物浓度。为了增加检测灵敏度，扩增SPRI使用夹心测定形式和近红外量子点（QDs），以增加信号强度。后直接SPRI检测重组生长激素中加标人血清中，SPRI信号由顺序注射检测抗体的涂有近红外量子点（纳米SPRI）扩增。在这项研究中，直接和扩增SPRI的诊断潜力进行了评估，用于测量的rhGH掺入人血清中和相比，直接用市售的ELISA试剂盒的功能。

SPRI和纳米单层屋面（单层屋面采用NanoEnhancers）的性能与ELISA法比较了重组人生长激素在复杂环境下的检测。在这些方法的设置的不同之处进行简单说明。对于SPRI（直接检测，图1），所述捕获抗体固定在表面上，然后将样品注射和分析物结合到所述传感器表面是实时和无标记的方式直接测量。然而，随着纳米SPRI（图1），之后所述分析物结合到传感器表面，一个连续的喷射之后是用涂有抗体检测扩增SPRI信号量子点。 <img alt="图1" src="/files/ftp_upload/53508/53508fig1.jpg" /> 图1.对比检测重组人生长激素的在C直接模式（单层屋面）和放大模式（纳米单层屋面）的示意图<html>粗鲁样品。配体（重组人生长激素或IgG特异性抗体）被固定在上SPRI生物芯片的阵列格式。靶蛋白（重组人生长激素）在人血清引入到传感器表面尖刺直接检测（单层屋面），依次突出了NanoEnhancers（纳米单层屋面）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 如用于ELISA，所述多孔板到达时已经预官能化的捕捉抗体，然后样品被引入，感兴趣的分析物将结合。甲检测抗体被引入然后添加底物。的光密度，然后在450nm处测量的。在这项研究中，商业ELISA试剂盒（ 图2）被用来测量生长激素掺入在10％的人血清。 8 / 53508fig2.jpg"/&gt; 图2的ELISA测定法过程的示意图 。重组人生长激素蛋白（蓝色椭圆）被引入到孔中已预先官能用单克隆抗体（黄色）到特定的rhGH。非特异性相互作用是通过漂洗用洗涤缓冲液的孔中，接着通过导入prefunctionalized与辣根过氧化物酶（HRP，紫色）检测抗体的消除。加入底物四甲基联苯胺（TMB，金）后，颜色的解决方案将改变。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3表示的rhGH的滴定曲线中掺入10％的人血清和绘制在获得在450nm处的OD。良好的线性响应，观察和检测限确定为1纳克/毫升。 VAR系数iation（CV）为6.5％，表明重复性好。 <img alt="图3" src="/files/ftp_upload/53508/53508fig3.jpg" /> 重组人生长激素图3. ELISA数据分析，浓度，血清飙升绘制在获得OD 450nm处。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 接下来，重组人生长激素的检测掺入人血清中进行了评估以SPRI。直接检测重组人生长激素的产生与相应的浓度梯度曲线（图4），每个点代表的反射率差从三个SPRI动力学曲线对于每个浓度计算得出的平均值。检测限（LOD）的极限被确定为3.61纳克/毫升。在SPRI直接检测分析法是高度可再现作为测定的CV只有4.1％。 <img alt="图4" src="/files/ftp_upload/53508/53508fig4.jpg" /> 图4.直接SPRI检测hGH的掺入在10％人血清，注射不同量的hGH的后所得归SPRI动力学情节人血清中，随后通过注射高盐缓冲液（垂直虚线）以除去非掺入特异性相互作用。代表不同量的hGH的结合了浓度梯度曲线人类血清已prefunctionalized与生物素化的hGH-特异性抗体的传感器表面尖刺。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 为了增加SPRI生物传感器的灵敏度，在NanoEnhancers（量子点预官能化的检测抗体）被依次troduced到传感器表面，以突出的rhGH的存在下掺入人血清。背景扣除后，所述NanoEnhancers能够然而扩增生物传感器响应提高到7.9％，以最小的信号变化（免疫球蛋白G（IgG）的特异性抗体，在反射率0.38％的变化;图5上的兴趣的成像的控制区。传感器表面透露，感兴趣的有重组人生长激素特异性抗体只有固定的区域经验，最大对比度变化直接与运动传感响应佐证。 <img alt="图5" src="/files/ftp_upload/53508/53508fig5.jpg" /> 图5.使用夹心测定法中掺入10％的人血清。SPRI动力学积于缓冲注射生长激素（30毫微克/毫升）的后（10毫米的PBS，150mM氯化钠，pH值= 7.4）到预检测重组人生长激素 -functionalized芯片采用11巯基烷酸/ rhGH-特异性抗体和IgG的控制-抗体，然后用BSA封闭，随后加入的检测抗体包被的量子点（NanoEnhancers）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 为了证明纳米SPRI生物传感器的实用性，重组人生长激素的粗样品在测量范围内进行了评估。延长的工作范围从30000微克/毫升至0.25微克/毫升的结果作为对加法NanoEnhancers（图6）的一个响应。值得注意的是，该滴定曲线上的每个点是从三次独立实验的平均值。因此，检测的下限计算为9.20微克/毫升和变异系数为20％。 <img alt="图6" src="/files/ftp_upload/53508/53508fig6.jpg" /> 图6。纳米SPRI检测hGH的尖刺人血清中。hGH_specific_Anti -量子点放大信号，人血清样品的归一化SPRI动能情节表示添加不同浓度的hGH的。一条垂直虚线（灰色）表示运行缓冲液的注入点。 （二）表示NanoEnhancers（hGH_specific_Anti-量子点）的注射不同量的hGH的后的结合了浓度梯度曲线掺加在人血清中于已被prefunctionalized与11-巯基十一酸/ hGH的特异性抗体的传感器表面。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig6large.jpg" target="_blank">请这里查看该图的放大版本。</a> 接着，发生在SPR反射率曲线作为浓度的函数的变化的直接和NanoEnhancer生物测定法（图7）之间进行比较。对于直接去25和0.25微克/毫升之间tection技术中，信号开始高原，这并不奇怪，因为这些浓度落在3纳克/毫升（图7）低于检测限。同样，对于扩增的技术中，低于9.2皮克/毫升信号检测限浓度开始高原和显示几乎没有变化。 <img alt="图7" src="/files/ftp_upload/53508/53508fig7.jpg" /> 图7. SPRI与纳米单层屋面的比较分析。这条线图描绘了反射率（％R）引入重组人生长激素后，接着注入NanoEnhancers（扩增检测）30000的百分比变化飙升人血清（直接检测）皮克/毫升，250微克/毫升，25微克/毫升，2.5微克/毫升和0.25微克/毫升。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p class="jove_content" fo:keep-together.within-page ="1"&gt;此外，纳米SPRI生物传感器的特异性进行了评估。胰岛素样生长因子-1（IGF-1）的人血清中掺入注射作为对照，因为刺激的hGH的IGF-1的分泌通过在肝细胞膜生长激素受体。在注入的IGF-1的血清后，将NanoEnhancers依次注入和SPR信号响应并没有表现出任何特异性结合（ 图8）。然而，当重组人生长激素飙升血清样品被注入到功能化重组人生长激素的抗体非常相同的地方，信号增强了观察。总之，纳米单层屋面平台已经展现了出色的特异性和选择性的重组人生长激素。 <img alt="图8" src="/files/ftp_upload/53508/53508fig8.jpg" /> 图8的纳米SPRI朝向的rhGH选择性评价。s中注入的rhGH（黑色）和IGF-1（红色）后的纳米SPRI生物传感器响应缟血清。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 使用皮尔逊相关系数来确定SPRI信号强度和ELISA光密度值（图9）之间的相关性执行的相关性分析。 p值&lt;0.01被认为是显著。如该图中图中，有重组人生长激素水平尖刺人血清SPRI和ELISA测量之间的良好的相关性。 r值是0.9263 9不同的样品。 <img alt="图9" src="/files/ftp_upload/53508/53508fig9.jpg" /> ELISA和纳米SPRI之间 图9. Pearson相关分析的曲线图相关联的纳米SPRI信号强度（y轴）采用ELISA光密度（x轴）的值。 （梨相关系数N = 9，R = 0.9263，P = 0.00000183）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53508/53508fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 平衡解离常数（K D）的用GraphPad软件用于ELISA测定。计算出的杀敌值约为79纳米（见表3）。在上（K a）和关闭（K D）率无法通过ELISA来确定。然而，使用数据分析软件，直接检测方法的结果与23日下午使用的22 kDa的对应于一个重组人生长激素分子的分子质量杀敌值。的开启和关闭速率计算为6.1×10 7 M -1 秒 -1，1.33×10 -3秒-1。这在本质上转换每秒的重组人生长激素和抗体复合物衰变的0.13％。作为一个mplified SPRI实验，NanoEnhancers和重组人生长激素作为计算的结合亲和力之间观察到更强的整体相互作用测定为4.3 PM。此外，更强的结合速率是观察NanoEnhancers和重组人生长激素比捕获抗体/生长激素，但解离速率表明，每秒NanoEnhancer /生长激素/捕获抗体衰变0.26％。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 方法 </td><td> 杀敌 （亲和） </td><td> K A（结合速率） </td><td> 杀敌 （离速率） </td><td> LOD </td></tr><tr><td> ELISA </td><td> 79.45×10 -9 M的 </td><td> NA </td><td> NA </td><td> 1ng / ml的</td></tr><tr><td> SPRI </td><td> 23.2×10 -12中号</td><td> 6.1×10 7 M -1 秒 -1 </td><td&gt; 1.33×10 -3 秒 -1 </td><td> 3.61纳克/毫升</td></tr><tr><td>纳米SPRI </td><td> 4.33×10 -12中号</td><td> 7.54×10 8 M -1 秒 -1 </td><td> 2.62×10 -3 秒 -1 </td><td> 0.0092纳克/毫升</td></tr></tbody></table>的亲和力的表3全动力学数据分析。评价，结合速率和解离速率使用GraphPad（ELISA）和数据分析（SPRI和纳米SPRI）软件的抗体反应。的亲合力使用的22 kDa的对应于一个重组人生长激素分子的分子量的确定被选择。检测限被确定为使用Excel的所有三项研究。

Watch this Scientific Journal Video about Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays at JoVE.com

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

所提出的工作（SPRI）测定法，特别是用于在加标人血清重组人生长激素的检测，通过比较SPRI直接结果与市售的酶联免疫吸附试验评估直接和扩增表面等离子体共振成像的诊断潜力（ELISA）套件。

人生长激素测定血清SPRI，纳米单层屋面和酶联免疫吸附测定比较分析

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

comparative-analysis-human-growth-hormone-serum-using-spri-nano-spri

Nanyang Technological University

Research

JoVE Journal

Biology

16.4K 次观看.  University of North Carolina at Greensboro. 所提出的工作（SPRI）测定法，特别是用于在加标人血清重组人生长激素的检测，通过比较SPRI直接结果与市售的酶联免疫吸附试验评估直接和扩增表面等离子体共振成像的诊断潜力（ELISA）套件。 

视频: Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

科研

癌症研究

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 ...

医学

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

化学

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

生物工程

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

免疫与感染

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

用超常光透射法定量测定人血清中的心脏标志物

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

Detection and Quantification of Calcitonin Gene-Related Peptide CGRP in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human plasma.

使用改良的酶联免疫吸附测定法检测和定量人血浆中的降钙素基因相关肽 （CGRP）

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a ...

生物化学

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 ...

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 ...

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 ...

人生长激素测定血清SPRI，纳米单层屋面和酶联免疫吸附测定比较分析

摘要

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此视频中的章节

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

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

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

对于血液处理快速的方法，以提高血浆肽水平的人血产量

用超常光透射法定量测定人血清中的心脏标志物

使用改良的酶联免疫吸附测定法检测和定量人血浆中的降钙素基因相关肽（CGRP）

人胰岛自身抗体的电化学发光测定

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

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

人生长激素测定血清SPRI，纳米单层屋面和酶联免疫吸附测定比较分析

摘要

探索更多视频

此视频中的章节

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

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

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

对于血液处理快速的方法，以提高血浆肽水平的人血产量

用超常光透射法定量测定人血清中的心脏标志物

使用改良的酶联免疫吸附测定法检测和定量人血浆中的降钙素基因相关肽 （CGRP）

人胰岛自身抗体的电化学发光测定

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

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

使用改良的酶联免疫吸附测定法检测和定量人血浆中的降钙素基因相关肽（CGRP）