双色抗原微阵列产生的IgG和IgM自身抗体的同时检测

摘要

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.

摘要

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.

引言

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 erythematosus^4-6. 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.

研究方案

1.稀释抗原和生成抗原微阵列

1. 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。
2. 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）。
3. 盖源板箔片和冷冻在-80°C，直到准备打印阵列。
4. 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。
5. 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。
6. 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。
7. 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。
8. 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。
9. 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。

2.探测抗原基因芯片与血清稀释

1. 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。
2. 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℃。
3. 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。
4. 带粘接膜覆盖，并于4℃用摇动孵育1小时。
5. 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。
6. 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。
7. 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。
8. 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。
9. 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。
10. 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。
11. 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。
12. 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。
13. 放置在不透光的盒滑动，直到准备好进行扫描。

3.扫描抗原微阵列和导出数据

1. 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。
2. 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。
3. 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）。
4. 加载滑待分析成通过使用"文件"按钮微阵列的软件。
5. 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。
6. 将在扫描图像阵列模板，以便阵列特征密切模板匹配越好。
7. 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。
8. 使用"文件"按钮将这些结果导出为文本文件。

4.使用微阵列显着性分析分析阵列数据（SAM）

1. 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。
2. 计算平均值的重复功能。
3. 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。
4. 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。
5. 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<0.05）。
6. 使用聚类和热图生成软件制作图像供呈现^8。

结果

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?...

讨论

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:10...

材料

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