(627b) Phi – a Parameter for Quantifying the Specificity of Post-Translational Modification Site Targeting Antibodies

Protein post-translational modifications (PTMs) are enzymatically-catalyzed alterations in amino acid side chains. PTM is a major mechanism for reversible regulation of protein activity, localization, and turnover in cells. Failure to balance the extent of modifications at critical regulatory sites have been identified in diseases ranging from cancer to immunological, endocrine, cardiovascular, and neurodegenerative disorders. For protein phosphorylation, the most common form of PTM, thousands of antibodies are currently available, many of which are being developed for clinical assays and as biotherapeutics. However, a quantitative measure of specificity for these antibodies, equivalent to dissociation constant for affinity, currently does not exist. Here we report a robust flow cytometry assay using human embryonic kidney (HEK) that enables the determination of a specificity parameter termed phi, that measures the fraction of non-specific signal in antibody binding. We apply this approach in validation of antibodies targeting site-specific phosphorylation sites in the human microtubule-associated protein tau. We validate our assay using 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) which have been used in hundreds of previous studies. We successfully determined the phi 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 phi values near 1, which indicates no detectable non-specific binding. AT270 showed phi 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 both in HEK cells and mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted in setting a standard and comparing the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general.