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 border="0" cellpadding="0" cellspacing="0" width="1054">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Ribosomal P0</td>
			<td style="width:225px;">Diarect</td>
			<td style="width:304px;">14100</td>
			<td style="width:240px;">dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human IgG</td>
			<td>Jackson Immuno</td>
			<td>009-000-003</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human IgM</td>
			<td>Jackson Immuno</td>
			<td>009-000-012</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse IgG</td>
			<td>Sigma-Aldrich</td>
			<td>I5381</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse IgM</td>
			<td>Biolegend</td>
			<td>401601</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double-stranded DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D1626</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">single-stranded DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D8899</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microarrayer</td>
			<td>Virtek</td>
			<td>VersArray Chipwriter Pro</td>
			<td>many types of arrayers are suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">solid printing pins</td>
			<td>Arrayit Corporation</td>
			<td>SSP015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">software for robotic microarrayer</td>
			<td>Virtek</td>
			<td>Chipwriter Pro&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST slides (2 Pad)</td>
			<td>GVS Northa America</td>
			<td>10485317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST frame</td>
			<td>GVS Northa America</td>
			<td>10486001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST incubation chambers (2 Pad)</td>
			<td>GVS Northa America</td>
			<td>10486242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384 well plates</td>
			<td>Whatman</td>
			<td>7701-5101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plate sealers</td>
			<td>VWR</td>
			<td>60941-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">foil plate covers</td>
			<td>VWR</td>
			<td>60941-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum</td>
			<td>Invitrogen</td>
			<td>12483020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Microarray Scanner</td>
			<td>Molecular Devices</td>
			<td>Axon 4200A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microarray software</td>
			<td>Molecular Devices</td>
			<td>Genepix 6.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clustering software</td>
			<td>eisenlab.org</td>
			<td>Cluster 3.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heatmap software</td>
			<td>eisenlab.org</td>
			<td>Treeview 1.60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microarray statistical software</td>
			<td>Stanford University</td>
			<td colspan="2">SAM 4.0 (Significance Analysis of Microarrays)</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.7K 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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

在乳腺癌检测人类白细胞抗原生物标志物的利用无标记生物传感技术

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

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 ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

从正式固定组织检测利沙病毒抗原的免疫化学测试

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...