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Abstract The ability to measure total and phosphorylated tau levels in clinical samples is transforming the detection of Alzheimer’s disease (AD) and other neurodegenerative diseases. In particular, recent reports indicate that accurate detection of low levels of phosphorylated tau (p-tau) in plasma provides a reliable biomarker of AD long before sensing memory loss. Therefore, the diagnosis and monitoring of neurodegenerative diseases progression using blood samples is becoming a reality. These major advances were achieved by using antibodies specific to p-tau as well as sophisticated high-sensitivity immunoassay platforms. This review focuses on these enabling advances in high-specificity antibody development, engineering, and novel signal detection methods. We will draw insights from structural studies on p-tau antibodies, engineering efforts to improve their binding properties, and efforts to validate their specificity. A comprehensive survey of high-sensitivity p-tau immunoassay platforms along with sensitivity limits will be provided. We conclude that although robust approaches for detecting certain p-tau species have been established, systematic efforts to validate antibodies for assay development is still needed for the recognition of biomarkers for AD and other neurodegenerative diseases. 

Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTM-targeting antibodies have reported frequent non-specific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phospho-threonine 231 (pT231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinity-matured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved > 20-fold over that of the wild-type antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity towards the non-phosphorylated target site, but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phospho-tau antibody. In cell- and tissue-imaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTM-specific antibodies, and highlight the importance of assessing the specificity in the antibody engineering process. 

Abstract 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. imageCover Image for this issue: doi:10.1111/jnc.14727.