TY - JOUR T1 - High specificity of widely used phospho-tau antibodies validated using a quantitative whole-cell based assay JF - bioRxiv DO - 10.1101/612911 SP - 612911 AU - Dan Li AU - Yong Ku Cho Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/04/18/612911.abstract N2 - 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 (HEK) 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 7 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 HEK 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.ADAlzheimer’s diseaseHEKHuman embryonic kidneyphospho-tauphosphorylated tauPHFpaired helical filamentEGFPenhanced green fluorescent proteiniRFPinfrared fluorescent proteinλPPlambda protein phosphataseWTwild-typeGSK-3βglycogen synthase kinase-3βPTMpost-translational modificationscFvsingle-chain antibody variable fragmentpThr231phospho-threonine 231RRIDResearch Resource Identifier ER -