Name: generation of two-color antigen microarrays for the simultaneous detection of igg and igm autoantibodies
Uploaded: 2016-09-15T13:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies at JoVE.com

A.C. was supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada and the Training Program in Regenerative Medicine (Canadian Institutes of Health Research). F.Y.Y.H. was supported by the Training Program in Regenerative Medicine. This work was funded by a grant from Astellas Pharma Canada. We also would like to thank Dr. Mark Menenghini (University of Toronto) for use of his Axon microarray scanner.

1.稀释抗原和生成抗原微阵列<ol><li>稀释在PBS中的抗原，以0.2毫克/毫升的最终浓度。打印一式两份可达162独特的抗原与9针的微阵列的配置。包括在抗原库作为阳性对照IgG和IgM的抗原。包括的PBS仅作为阴性对照。 </li><li>加入20微升的每种抗原到384孔源板。添加抗原到源盘在镜像打印头的设置团体（ 例如 ，安排抗原的9组时9引脚用于打印）。 </li><li>盖源板箔片和冷冻在-80°C，直到准备打印阵列。 </li><li>通过在超声处理浴使用去离子水将其培养为3×1分钟清洗固体微阵列引脚。在机架的地方针脚干燥，然后安排打印头芯片的引脚。对于9针，使用3×3的配置。 </li><li>对于打印程序微阵列通过设置打印头数针运行，幻灯片的数量来打印，垫片的数目在每个滑动，并为每个抗原重复点的数目。通常情况下，使用9销70滑动，第2焊盘/滑动，并为每个抗原2重复点。计划到微阵列销超声处理在水中抗原的不同群体之间。 </li><li>解冻源盘和在100×g的1分钟，然后离心板。将源盘在微阵列指定地点。除去被覆盖要打印的抗原的第一组的箔的部分。 </li><li>排列在表面点样仪和运行打印程序未打印幻灯片。在室温打印幻灯片与湿度对点样仪设定在55 - 60％，而阵列机加湿器构建。 </li><li>每一组抗原被印在所有的幻灯片后，暂停微阵列。覆盖只是印有铜箔的抗原（防止蒸发），并发现要打印抗原的下一组。继续打印程序。 </li><li>毕竟抗原已打印，覆盖源PL吃了在-80℃的新的金属箔片和冻结。将在滑动盒和真空密封打印幻灯片。载玻片可使用第二天或长达一个月后。 </li></ol> 2.探测抗原基因芯片与血清稀释<ol><li>将滑入使用培养室帧。加入700微升封闭缓冲液（2.5％[体积/体积]胎儿小牛血清（FCS），0.1％[体积/体积]吐温20在PBS中）的每个阵列表面。 </li><li>放置在框架和地点粘合膜在用一块湿纸巾的密封容器中。在摇杆孵育O / N在4℃。 </li><li>在封闭缓冲液100:稀释血清样品1。吸阻塞来自阵列溶液，并添加500μl的稀释样品至每个阵列的表面上。 </li><li>带粘接膜覆盖，并于4℃用摇动孵育1小时。 </li><li>稀释的二抗在封闭缓冲液。对人类的研究，稀一个Cy3标记的山羊抗 - 人IgG抗体以0.33微克/毫升和Cy5标记的山羊抗 - 呼玛Ñ​​的IgM抗体至0.25微克/毫升。对小鼠的研究中，稀一个Cy3标记的山羊抗小鼠IgG抗体至0.38微克/毫升和Cy5标记的山羊抗小鼠IgM抗体至0.7微克/毫升。 </li><li>从阵列吸的样品和冲洗阵列表面4次漂洗缓冲液（在PBS中的0.1％吐温20）。倒入缓冲到到幻灯片和快速轻弹关闭。 </li><li>加入700μl封闭液与摇摆洗每个阵列表面和孵化10分钟，在室温中。重复洗涤步骤两次。 </li><li>加入500μl稀释的第二抗体与各滑动表面并与粘合剂膜覆盖。孵育45分钟，在4℃下用摆动。 </li><li>从幻灯片吸二级抗体混合物作为冲洗上述4倍。洗涤滑动用封闭/稀释缓冲液700微升如上3次。 </li><li>除去从浸渍在PBS中的金属滑动机架帧和地方幻灯片。孵育在室温20分钟以轨道振荡。在PBS的新容器中，并培育另一个20米在发抖。 </li><li>地方滑动架中的去离子水15秒的容器。安置机架在水的新容器和孵化另一个15秒。 </li><li>要干片，离心机和旋转220 XG ELISA板适配器，在室温下5分钟的地方滑架上。 </li><li>放置在不透光的盒滑动，直到准备好进行扫描。 </li></ol> 3.扫描抗原微阵列和导出数据<ol><li>扫描载玻片使用​​微阵列扫描仪，可以检测Cy3和Cy5的荧光信号。为了调整扫描器的光电倍增管（PMT）的水平，预扫描只与二次抗体探测的幻灯片。 注:该幻灯片应该有印有包括正（IgG和IgM）的全部抗原库以及阴性（PBS）的控制。预扫描是低分辨率扫描，允许用户迅速地找到最佳的PMT设置。 </li><li>设置PMT值，以便人IgG等功能（详见Cy3的信道）和嗡嗡声IgM的功能（在Cy5的通道）也有类似的中位数荧光强度减去背景（MFI-B）（通常为40,000）。 PMT中水平通过按下"硬件"按钮组。一旦设定，保持这些PMT设置不变。 </li><li>通过按下"扫描"按钮扫描上的两个通道中的实验滑动。每次扫描后保存幻灯片图像（两个通道）。 </li><li>加载滑待分析成通过使用"文件"按钮微阵列的软件。 </li><li>通过使用"文件"按钮加载基因数组列表（GAL）文件。在GAL文件具有与阵列特征的身份阵列的布局。 </li><li>将在扫描图像阵列模板，以便阵列特征密切模板匹配越好。 </li><li>对齐中的所有块的特征与通过按下"调整"按钮的模板。该计划通常会发现的圆形特征的轮廓，但一些手动调整可能是必要的。这些调整可以通过输入功能模式进行。然后软件将每个抗原的计算MFI-B。 </li><li>使用"文件"按钮将这些结果导出为文本文件。 </li></ol> 4.使用微阵列显着性分析分析阵列数据（SAM） <ol><li>加载单独的文本文件转换成Excel和确定具有MFI-B的Cy3和Cy5通道列。 </li><li>计算平均值的重复功能。 </li><li>对于任何负面的MFI-B取代除以100原始MFI-B，然后通过计算日志基座2变换数据与10分的负数。 </li><li>如前面描述的7与分析微阵列（SAM）的意义分析日志转换后的数据。 </li><li>使用两个群体（ 例如 ，健康对照与患者），标记一组"1"和该组的研究"2"。使用SAM两班，不成对的分析来识别抗原的反应性是有s两组ignificantly不同（Q值&lt;0.05）。 </li><li>使用聚类和热图生成软件制作图像供呈现8。 </li></ol>

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The authors have nothing to disclose.

这里所描述的协议允许使用抗原微阵列技术的自身抗体的定量。抗原芯片提供比传统的ELISA几个优点筛选自身抗体。首先，多种抗原包括核酸，蛋白质，肽和细胞裂解物的可以排列到硝化纤维素包被的载玻片，因此允许自身抗体的多路复用筛选。另外，由于抗原的纳升被点样到每个幻灯片只有抗原的微克是必要的，以产生阵列。该阵列还需要很少的患者样本，因为只需要5微升血清在1探测阵列面:100样品稀释。最后，阵列点样到硝化纤维素已被证明是在筛选抗体9比常规的ELISA更敏感。 除了使用抗原微阵列来筛选抗体，该阵列可以是使用d，来检测对非自身蛋白质（ 例如病毒蛋白）的反应性，以限定的单克隆抗体的特异性，如先前9-12描述测量移植非HLA抗体的水平。抗原微阵列也可以用于执行详细的表位作图实验，其中一个单一的蛋白的重叠肽被排列到滑动。最近的一项研究中使用这种方法来定义在系统性红斑狼疮13自身抗体的目标U1-70K蛋白的抗原表位。 使用此处描述的协议，IgG和IgM的反应性的检测是在宽范围的血清稀释的线性。使用阳性对照血清与已知的活性核糖体P抗原和单链DNA系统性红斑狼疮病人进行这些研究。 IgM的反应性的单链DNA是在1血清稀释线性:125至1:32,000，对应于17000〜50的IgG的反应性，以MFI-B的核糖P是1血清稀释线性:4000至1:512,000，对应于32000〜200的MFI-B在稀释低于1:4000（和MFI-B的超过32,000以上），IgG的反应性变平的检测曲线而不再是线性的。在心脏移植受者的工作，血清常规地稀释至1:100的MFI-B为抗原在这种稀释很少超过10,000。用于与患者的自身免疫性疾病具有高滴度抗体的研究中，患者样品可能需要进一步稀释，以确保大多数自身抗体的反应性的落测定的线性范围内。 而MFI-B可以是对患者组之间的自身抗体的反应性比较是有用的，它也可能是有用的这个值转换成自身抗体水平的更定量测量。这可以通过点样纯化的IgG和IgM的多个已知稀释到幻灯片，以产生标准曲线来实现。使用该曲线，MFI-B然后可以跨形成为对于每个抗原的自身抗体的水平。 在这里介绍的协议中，使用2-垫硝化纤维素包被的载玻片。这些幻灯片，两个相同的数组，可以用两个单独的血清样品进行探测，被印刷到每个幻灯片。这允许来自同一患者（如前和后处理）两个样品要在同一个幻灯片，最大限度地减少interslide变性进行比较。三十二个载玻片用64个体患者样品在一天同一时间例行处理。最新的阵列上印有9针，允许162抗原一式两份进行打印（每个引脚输出相当于18抗原重复打印了36功能子阵列）。 在这个协议产生的抗原微阵列印有固体微阵列引脚。带实心销印刷耗费更多的时间比用鹅毛笔风格针打印，因为微阵列必须重新浸入后，各t源板IME使销触摸滑动面。因为它们产生高度可重复的，近圆形阵列功能（约500微米直径）实心针进行使用。这些特征可以容易地检测并通过扫描仪软件所概述的，并且需要最小的手动调整量化阵列的特性。实心销也允许细胞裂解物要被打印，这可能是难以与主轴销打印由于其较高的粘度。由于涉及用固体销长打印时间（有时10 - 11小时的打印70幻灯片），即不被打印在源板抗原与箔覆盖以防止蒸发。 这里所描述的协议呈现给同一滑动面上分离IgG和IgM的反应性的方法。阵列已被优化以筛选用对标记具有不同的荧光团的第二抗体的人和小鼠抗体。关键这两个颜色的方法是确定塞科是高度特异性的任一IgG或IgM同种型ndary抗体。所使用的二次抗体识别IgG或IgM的Fc部分，并且不彼此交叉反应。分离IgG和IgM信号鉴于免疫应答这些抗体的不同的功能是重要的。例如，IgM是在上级B1细胞产生，并且通常的早期免疫反应的标志。另一方面，IgG的是后来的免疫反应，其发展为类切换14的结果的标记。通过分别检测IgG和IgM的信号，它也可能识别患者样品中的IgM类风湿因子（ 即 ，结合于IgG IgM的免疫球蛋白已发现到阵列）。通过发现抗体的不同部分（ 例如 ，Fc的抗的Fab），不同IgG抗体的表位的类风湿因子的结合可以量化。用扫描仪具有多于两个激光器的可用性，另外的第二进制抗体可以被添加到当前的辅助抗体对识别的IgA，IgD的或IgE抗体的结合。第二抗体也可以识别出将允许绑定到抗原在单个阵列上独立的IgG亚类（IgG1的，IgG2的，IgG3的，IgG4的）鉴定。 最后，该抗原微阵列技术是一种强大的技术允许血清自身抗体的高通量鉴定。这种技术是高度可定制的和敏感的。基于荧光检测系统已为人类和小鼠的研究得到了优化，并允许IgG和IgM抗体的同时检测。

Autoantibodies are present in many disease states and can often have direct pathogenic activity1. Identification of autoantibodies is important for diagnosis of certain diseases, for prognosis of disease outcome, and for the classification of patients who may benefit from specific therapies2. Autoantibodies are typically identified in patient serum using the ELISA technique; however, screening for multiple antigens with this technique is laborious and consumes a large quantity of patient sample. New technologies are therefore needed to profile autoantibodies on a larger scale.
The antigen microarray technique is a proteomic technology that allows autoantibodies to be profiled in a multiplex fashion3. In the first step of this process, an antigen library is arrayed onto a slide surface using a robotic microarrayer. The slides are probed with diluted serum and then fluorescent-labeled secondary antibodies are added. Antibody reactivities are visualized by scanning the slides with a microarray scanner and quantified by fluorescent intensities. Antigen microarrays offer multiple advantages over the ELISA technique in screening for autoantibodies: 1) they require only microliters of serum to profile autoantibodies to multiple antigens simultaneously, 2) they use antigen sparingly, as only nanoliters of antigen are spotted onto the arrays, 3) they have enhanced sensitivity3 compared to ELISA and 4) they allow for the simultaneous yet, separate detection of more than one antibody isotype. Antigen microarrays have been used to profile autoantibodies in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus4-6. In all three of these diseases, new insight into disease pathogenesis was obtained from profiling autoantibodies on a large scale with the arrays. 
Here we describe a protocol to generate antigen microarrays using nitrocellulose-coated slides. A variety of antigens including proteins, peptides, and cell lysates can be arrayed onto the slides using this technique. The arrays can be easily customized by including antigens of interest in the source plate that holds the antigen library. In addition, we show how a pair of secondary antibodies can be used to separate IgG and IgM reactivities on the same slide surface. We have now optimized this technique to measure autoantibodies in both humans and mice.

<table><tbody><tr><td>Ribosomal P0</td><td>Diarect</td><td>14100</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>human IgG</td><td>Jackson Immuno</td><td>009-000-003</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>human IgM</td><td>Jackson Immuno</td><td>009-000-012</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>mouse IgG</td><td>Sigma-Aldrich</td><td>I5381</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>mouse IgM</td><td>Biolegend</td><td>401601</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>double-stranded DNA</td><td>Sigma-Aldrich</td><td>D1626</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>single-stranded DNA</td><td>Sigma-Aldrich</td><td>D8899</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>microarrayer</td><td>Virtek</td><td>VersArray Chipwriter Pro</td><td>many types of arrayers are suitable</td></tr><tr><td>solid printing pins</td><td>Arrayit Corporation</td><td>SSP015</td><td></td></tr><tr><td>software for robotic microarrayer</td><td>Virtek</td><td>Chipwriter Pro&amp;nbsp;</td><td></td></tr><tr><td>FAST slides (2 Pad)</td><td>GVS Northa America</td><td>10485317</td><td></td></tr><tr><td>FAST frame</td><td>GVS Northa America</td><td>10486001</td><td></td></tr><tr><td>FAST incubation chambers (2 Pad)</td><td>GVS Northa America</td><td>10486242</td><td></td></tr><tr><td>384 well plates</td><td>Whatman</td><td>7701-5101</td><td></td></tr><tr><td>plate sealers</td><td>VWR</td><td>60941-062</td><td></td></tr><tr><td>foil plate covers</td><td>VWR</td><td>60941-124</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Fetal calf serum</td><td>Invitrogen</td><td>12483020</td><td></td></tr><tr><td>Cy3 goat anti-human IgG</td><td>Jackson Immuno</td><td>109-165-096</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy5 goat anti-human IgM</td><td>Jackson Immuno</td><td>109-175-129</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy3 goat anti-mouse IgG</td><td>Jackson Immuno</td><td>115-165-071</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy5 goat anti-mouse IgM</td><td>Jackson Immuno</td><td>115-175-075</td><td>use working stock in 50% glyercol</td></tr><tr><td>Microarray Scanner</td><td>Molecular Devices</td><td>Axon 4200A</td><td></td></tr><tr><td>Microarray software</td><td>Molecular Devices</td><td>Genepix 6.1</td><td></td></tr><tr><td>Clustering software</td><td>eisenlab.org</td><td>Cluster 3.0</td><td></td></tr><tr><td>Heatmap software</td><td>eisenlab.org</td><td>Treeview 1.60</td><td></td></tr><tr><td>Microarray statistical software</td><td>Stanford University</td><td>SAM 4.0 (Significance Analysis of Microarrays)</td><td></td></tr></tbody></table>

generation of two-color antigen microarrays for the simultaneous detection of igg and igm autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

抗原是由机器人微阵列布置在384孔板和印刷到载玻片如图1， 图2示出了放置在培养室和处理后的扫描滑动一帧滑动。 图3示出了阳性和阴性对照载玻片。负滑动仅与二级抗体探测，与阳性对照载玻片从系统性红斑狼疮的患者探测血清。通过使用两个单独标记的二次抗体，IgG和IgM的反应性可以在同一滑动表面上分离。 图4示出了针对单链DNA（ssDNA）和IgG在宽针对核糖体的P（日波P）的抗体的IgM抗体的荧光强度范围血清稀释的。在从与反对瑞博P已知的反应性系统性红斑狼疮患者的这个实验阳性对照血清使用。林耳响应的IgM抗体通道在所有血清稀释观察。线性响应于IgG的信道到约30,000的MFI-B的观察到的。这点之后，反应开始饱和并增加抗体日波P不会导致在MFI-B的相应增加。 图5示出的阵列是如何可用于检测人血清中的类风湿因子。在这项研究中，使用从患者用冷球蛋白血症血管炎与记录类风湿因子的血清的167国际单位/毫升（通常&lt;11国际单位/毫升）（IgM抗体抗IgG抗体）。使用的第二抗体单独，没有IgM的反应性被认为是针对被点样到阵列的抗体。与此相反，显著的IgM反应性（约5000的MFI B）的抗IgG的检测当滑动与从类风湿因子的患者血清探测。 图6示出了双色抗原微阵列用于与小鼠血清利用的发展。在该图中，小鼠IgM第在点样到阵列仅由到阵列仅由抗小鼠IgG第二抗体检测到有斑点的抗小鼠IgM次级抗体和小鼠IgG检测的图上的扫描的模板网格7示出对准使用微阵列分析软件阵列图像。一旦该阵列的功能已被与网格对齐，阵列特征进行分析，以获得用于每个特征值荧光强度减去背景。 <img alt="图1" src="/files/ftp_upload/54543/54543fig1.jpg" /> 图1.抗原微阵列。抗原的产生首先被放置在源板。在图中所示的源板，抗原被布置在9组，匹配打印头销的3×3布局。然后在源板被放置在机器人微阵列，然后将抗原点到的幻灯片。在这个例子中，2-垫FAST载玻片ARE中使用，允许同一阵列要看准到2硝化纤维表面。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54543/54543fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54543/54543fig2.jpg" /> 图2. 抗原微阵列。幻灯片的处理中使用的培养室放置在快速帧和通过添加样品，然后二级抗体进行处理。抗原反应性，揭示与能够检测荧光信号的扫描器。 2垫FAST幻灯片的扫描图像显示， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54543/54543fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54543/54543fig3.jpg" /> &lt;BR /&gt; 图3. 双色抗原微阵列优化与人类血清的使用。 （A）的IgG，IgM抗体，和IgG的Fc仅与第二抗体探测阵列上进行检测。这三种抗原是阳性对照，以显示第二抗体的特异性。红色荧光（IgM的信道）表示抗 - 人IgM第二抗体的结合。绿色荧光抗体（IgG通道）表示的抗人IgG第二抗体的结合。 IgG的Fc的抗原是由于过饱和白色。从已知的反应性瑞博P抗原系统性红斑狼疮患者的阳性对照血清探测阵列上检测到（B）有更多的反应性。的框表示，其中的DNA和日波P的抗原已被被排列的位置。（C）的阳性对照血清中的荧光强度的定量表明，大多数对DNA抗原的抗体反应性的是免疫球蛋白的中号同种型。（D）的阳性对照血清中的荧光强度的定量表明，大部分针对日波P抗原抗体反应性的是IgG同种型。阵列特征点样两份和直径约为500微米。图表显示平均值±标准差为阵列功能。双链DNA，双链DNA;单链DNA，单链DNA; MFI-B，中值荧光强度减去背景;核糖P，核糖体P。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54543/54543fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54543/54543fig4.jpg" /> 图4. 荧光强度与血清稀释因子（A）的MFI-B在宽范围的血清稀释度的示出了用于IgM的抗单链DNA和IgG抗日波P.对于这些实验中，使用用抗日波P和ssDNA的已知抗体反应性的阳性对照血清。 MFI-B被以大约65,000值饱和的。所述MFI B数据（B）的LOG2变换揭示在IgG和IgM通道都在宽范围的抗原反应性的线性响应。超过30,000 MFI-B，IgG的信号开始饱和，从而失去线性。阵列的功能包括在6个重复被发现。图表显示平均值±标准差为阵列功能。单链DNA，单链DNA; MFI-B，中值荧光强度减去背景;核糖P，核糖体P。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54543/54543fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54543/54543fig5.jpg" /> 图5. 类风湿因子的检测与双色抗原微阵列。 <str翁&gt;（A）的IgG，IgM抗体，和IgG的Fc仅与第二抗体探测阵列上进行检测。红色荧光（IgM的信道）指示抗IgM次级抗体的结合。绿色荧光抗体（IgG通道）指示抗IgG二级抗体的结合。 IgG的Fc的抗原是由于过饱和白色。（B）从具有高类风湿因子冷球蛋白血症血管炎患者血清探测阵列。 IgG的特征为绿色，黄色，由于患者的血清中的IgM结合固定的IgG。有趣的是，该患者也有IgM型抗IgG抗体为IgM抗体功能出现橙色。病人也有先天性心脏疾病的历史和指出具有高反应性的IgG和IgM只与二次抗体探测阵列上设有表明二级抗体没有心肌蛋白肌钙蛋白C.（C）的定量任何交叉反应性。（D）的定量的与病人血清探测阵列上的IgG和IgM特征证实类风湿因子的存在。特征点样两份和直径约为500微米。图表显示平均值±标准差为阵列功能。 MFI-B，中位数荧光强度减去背景。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54543/54543fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54543/54543fig6.jpg" /> 图6 的双色阵列发展到简介小鼠血清抗体:（A）阵列仅与第二抗体进行探测。红色荧光指示抗小鼠IgM二级抗体的结合。绿色荧光表示抗小鼠IgG二抗的结合。被点样到阵列的小鼠IgG仅由防谋检测本身IgG二抗，以及被发现仅由抗小鼠IgM二级抗体检测到的小鼠IgM。关于由该二级抗体检测出的阵列的其他功能是针对小鼠IgG或小鼠IgM捕获抗体。用抗组氨酸标记的小鼠IgG单克隆抗体探测（B）的阵列。在此阵列中，日波P抗原（重组his标记蛋白）是绿色，这表明它仅由抗小鼠IgG第二抗体进行检测。这是考虑到的单克隆抗体是IgG同种型的预期。当阵列单独第二抗体（橙色框）探测未检测中的核糖P抗原。IgG抗体，IgM抗体（三）定量和Ribo P与抗组氨酸标记的鼠IgG单克隆抗体探测阵列上的功能。阵列特征点样在六个重复和直径约为500微米。图表显示平均值±标准差为阵列功能。 MFI-B，中位数氟escence强度减去背景;核糖P，核糖体P。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54543/54543fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54543/54543fig7.jpg" /> 图7. 模板电网对准到一个数组图片，在这张截图，模板网格，这是从加仑文件生成的，与以前扫描的抗原分子微阵列排列。该阵列由系统性红斑狼疮和血液凝固障碍谁有许多自身抗体的反应性的患者的血清探测。绿色荧光代表的IgG抗体的结合和红色荧光代表IgM抗体结合。由实心销打印的特点是接近圆形，容易被微阵列软件（Genepix）中概述。一旦网格对齐完成后，softwarE能计算每个阵列功能的中位数荧光强度减去背景（在Cy3和Cy5通道两者）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54543/54543fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies at JoVE.com

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

双色抗原微阵列产生的IgG和IgM自身抗体的同时检测

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

generation-two-color-antigen-microarrays-for-simultaneous-detection

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

12.8K 次观看.  University Health Network. We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest. 

视频: Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

科研

生物工程

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 ...

免疫与感染

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

生物学

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

使用流感抗原微阵列测量病毒亚型血清抗体的广度

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

血清中 疏螺旋体特异性 IgG 和 IgM 的多重免疫检测

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

作者聚焦:扩大用于莱姆疏螺旋体病诊断和病原体研究的多重免疫检测的范围 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

在多重血清学检测中同时检测不同抗体类别

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

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

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

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

同时筛查1型糖尿病和多种自身免疫性疾病的高通量电化学发光7重检测

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

医学

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

双色抗原微阵列产生的IgG和IgM自身抗体的同时检测

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此视频中的章节

无交叉反应和无免疫多路复用夹芯芯片

人胰岛自身抗体的电化学发光测定

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

使用流感抗原微阵列测量病毒亚型血清抗体的广度

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别

同时筛查1型糖尿病和多种自身免疫性疾病的高通量电化学发光7重检测

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array