Specificity Controls for Immunocytochemistry: The Antigen Preadsorption Test Can Lead to Inaccurate Assessment of Antibody Specificity
Specificity Controls for Immunocytochemistry: The Antigen Preadsorption Test Can Lead to Inaccurate Assessment of Antibody Specificity
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DOI：
10.1369/0022155411434828
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发表时间：
2012-03-01
影响因子：
3.2
通讯作者：
Danbolt, Niels Christian
中科院分区：
文献类型：
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作者：
Holmseth, Silvia;Zhou, Yun;Danbolt, Niels Christian
生物医学研究界直接或间接依赖于免疫细胞化学数据。不幸的是，验证标记特异性是困难的。一种常见的专属性试验是预吸附试验。该试验旨在检测粗抗血清，但现在经常用于验证单克隆抗体和亲和纯化的多克隆抗体。在这里，作者评估了这个测试的力量。在样品上测试针对3种蛋白质（EAAT 3，slc 1a 1; EAAT 2，slc 1a 2; BGT 1，slc 6a 12）上的不同表位的9种亲和纯化抗体（组织切片和固定或不固定的蛋白质印迹）。所选抗体显示出一定程度的交叉反应性，如通过标记来自敲除小鼠的样品所定义的。作者表明，抗原预吸附阻断了野生型和敲除样品的所有标记，这意味着预吸附也阻断了与交叉反应性表位的结合。他们展示了这如何产生特异性的错觉，并说明了灵敏度-特异性关系，良好的阴性对照的重要性，固定可以产生新的表位，并且存在于切片中的交叉反应表位可能不存在于Western印迹中，反之亦然。总之，他们反对不加批判地使用预吸附试验，并在这样做的过程中，解决了一些与免疫细胞化学特异性试验相关的其他问题。（J Histochem Cytochem 60：174-187，2012）
The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing. (J Histochem Cytochem 60: 174-187, 2012)