High specificity of widely used phospho‐tau antibodies validated using a quantitative whole‐cell based assay

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 .