The 'reverse capture' autoantibody microarray: a native antigen-based platform for autoantibody profiling
The 'reverse capture' autoantibody microarray: a native antigen-based platform for autoantibody profiling
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DOI：
10.1038/nprot.2006.66
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Liu, Brian C-S
中科院分区：
文献类型：
--
作者：
Ehrlich, Joshua R.;Qin, Shuzhen;Liu, Brian C-S
我们之前曾报道过“反向捕获”自身抗体微阵列的开发和使用，用于抗原-自身抗体分析研究。我们开发了“反向捕获”自身抗体微阵列，使用户能够表征和比较自身抗体概况。基于 ELISA 的双抗体夹心免疫分析，我们的“反向捕获”方案有助于检测对天然宿主抗原的自身免疫。与传统蛋白质阵列相比，我们的方法的优势在于能够检测天然抗原翻译后修饰 (PTM) 上发现的表位的自身免疫。该方法的第一步是将天然抗原固定到阵列表面上点样的单克隆抗体上。使用微阵列捕获的抗原作为“诱饵”，然后将阵列与来自测试和对照样品的差异标记 IgG 一起孵育，并执行两片染色交换以标准化染料效果。在本协议中，我们详细描述了“反向捕获”自身抗体微阵列，该方法可以在 1-2 天内在 9-10 小时内完成。
We have previously reported the development and the use of a 'reverse capture' autoantibody microarray for studies of antigen-autoantibody profiling. We developed the 'reverse capture' autoantibody microarray to allow the user to characterize and to compare autoantibody profiles. Based on the dual-antibody sandwich immunoassay of ELISA, our 'reverse capture' protocol facilitates the detection of autoimmunity to native host antigens. Our method has the advantage over traditional protein arrays of being able to detect autoimmunity to epitopes found on the post-translational modifications (PTMs) of native antigens. The first step of this method is to immobilize native antigens onto the monoclonal antibodies spotted on the array surface. Using the antigens captured by the microarray as 'baits,' we then incubate the array with differentially labeled IgG from test and control samples, and perform a two-slide dye-swap to normalize for dye effects. In this protocol we present a detailed description of the 'reverse capture' autoantibody microarray, a method that can be completed in 9-10 h over 1-2 d.