Department of Biomedical Engineering, University of Connecticut, Storrs, Connecticut, USA.
Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, Connecticut, USA.
J Neurochem. 2020 Jan;152(1):122-135. doi: 10.1111/jnc.14830. Epub 2019 Sep 4.
Antibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies. In order to quantify and compare the specificity phospho-tau antibodies and other post-translational modification site-specific antibodies in general, a measure of specificity is urgently needed. Here, we report a robust flow cytometry assay using human embryonic kidney cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non-specific signal in antibody binding. We validate our assay using anti-tau antibodies with known specificity profiles, and apply it to measure the specificity of seven widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have Φ values near 1, which indicates no detectable non-specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non-specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non-specific binding of AT270 to non-tau proteins found in human embryonic kidney cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general. Cover Image for this issue: doi: 10.1111/jnc.14727.
针对微管相关蛋白 tau 的定义磷酸化位点产生的抗体被广泛用于科学研究，并应用于临床检测。然而，最近的研究揭示了这些抗体中存在令人震惊的非特异性结合程度。为了量化和比较磷酸化 tau 抗体和其他翻译后修饰位点特异性抗体的特异性，迫切需要一种特异性测量方法。在这里，我们报告了一种使用人胚肾细胞的稳健的流式细胞术检测方法，该方法能够确定一个特异性参数 Φ，该参数衡量抗体结合中非特异性信号的分数。我们使用具有已知特异性特征的抗 tau 抗体验证了我们的检测方法，并应用该方法测量了七种广泛使用的磷酸化 tau 抗体（AT270、AT8、AT100、AT180、PHF-6、TG-3 和 PHF-1）等的特异性。我们成功地确定了除 AT100 之外的所有抗体的 Φ 值，AT100 在我们的检测中没有显示出可检测的结合。我们的结果表明，抗体 AT8、AT180、PHF-6、TG-3 和 PHF-1 的 Φ 值接近 1，这表明没有可检测到的非特异性结合。AT270 显示的 Φ 值约为 0.8，这意味着大约 20%的结合信号来自非特异性结合。使用免疫细胞化学和蛋白质印迹的进一步分析证实了 AT270 与人胚肾细胞和小鼠海马体中非 tau 蛋白的非特异性结合。我们预计，这里引入的定量方法和参数将被广泛采用，作为报告磷酸化 tau 抗体特异性的标准，并且可能作为翻译后修饰靶向抗体的一般标准。本期的封面图片：doi: 10.1111/jnc.14727。
