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

Abstract

List of abbreviationsAD: Alzheimer's disease; HEK: Human embryonic kidney; phospho-tau: phosphorylated tau; PHF: paired helical filament; EGFP: enhanced green fluorescent protein; iRFP: infrared fluorescent protein; λ P P : lambda protein phosphatase; WT: wild-type; GSK-3β: PTM: post-translational modification; glycogen synthase kinase-3β; scFv: single-chain antibody variable fragment; pThr231: phospho-threonine 231, RRID: Research Resource Identifier. AbstractAntibodies 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. AT180 (pT231) EGFP-tau: WT iRFP-tau: WT (λPP) EGFP-tau (WT) iRFP-tau WT (λPP) EGFP-tau (WT) Ab binding iRFP-tau (T231A) iRFP-tau (T231A) Ab binding EGFP-tau (WT) EGFP-tau (WT) Ab binding Ab binding iRFP-tau WT (λPP) AT180 (pT231)

Expression of GFAP and Tau Following Blast Exposure in the Cerebral Cortex of Ferrets

“…A report using Western blot to evaluate the specificity of various pTau antibodies noted that AT8 showed a relatively high level of nonspecificity in their assay, whereas PHF1 and CP13 showed high specificity (60). Another recent study using a different assay (flow cytometry in an HEK binding assay) found that AT8 and PHF1 showed highly specific binding (61). These findings may help to explain the different patterns observed immunohistochemically with different pTau antibodies.…”

Tau forms oligomeric complexes on microtubules that are distinct from tau aggregates

“…However, the nature of tau present within these aggregates is unclear. In order to investigate the phosphorylation state of tau within these aggregate species, we performed twocolor SMLM using two commonly used phospho-tau antibodies, Thr231 and AT8, which are known to have high labeling specificity (32). Thr231 targets tau that is phosphorylated at the 231st threonine, which is considered to be an early marker of tau aggregation.…”

Tau nuclear translocation is a leading step in tau pathology process through P53 stabilization and nucleolar dispersion

“…We isolated primary cortical neurons from embryonic mice (E15‐16) and starved them for 7 DIV to investigate P‐tau's pathogenic intraneuronal spread (Figures 1 and S1). Then, we examined the formation and localization of cis P‐tau (early driver), AT8 P‐tau (early tangle), and AT100 P‐tau (late tangle) (Li & Cho, 2020) at various time points. Following starvation, we observed significant intraneuronal spread of P‐tau markers.…”