Development and Validation of a High Sensitivity Assay for Measuring p217 + tau in Cerebrospinal Fluid

Abstract

Background: Early and accurate detection and staging is critical to managing Alzheimer’s disease (AD) and supporting clinical trials. Cerebrospinal fluid (CSF) biomarkers for amyloid-β peptides, tau species, and various neurodegenerative and inflammatory analytes are leading the way in this regard, yet there is room for improved sensitivity and specificity. In particular tau is known to be present in many different fragments, conformations, and post-translationally modified forms. While the exact tau species that might best reflect AD pathology is unknown, a growing body of evidence suggests that forms with high levels of phosphorylation in the mid-region may be especially enriched in AD. Objective: Develop an assay for measuring p217tau in CSF. Methods: Here we describe the development and validation of a novel sELISA for measuring CSF tau species containing phosphorylation at threonines 212 & 217, aka p217 + tau, using the PT3 antibody. Results: While the analyte is present at extremely low levels the assay is sufficiently sensitive and specific to quantitate p217 + tau with excellent precision, accuracy, and dilution linearity, allowing good differentiation between diagnostic subgroups. The p217 + tau measurements appear to track AD pathology better than the commonly used p181tau epitope, suggesting superior diagnostic and staging performance. Finally, the assay can also be configured to differentiate antibody-bound versus antibody-free tau, and therefore can be used to measure target engagement by p217 + tau-targeting immunotherapeutics. Conclusion: The assay for measuring p217 + tau described here is highly sensitive, accurate, precise, dilution linear, and shows good potential for identifying and staging AD.

Development and validation of a high‐sensitivity assay for measuring p217+tau in plasma

“…The assay relies on the high affinity and specificity of the PT3 mAb, which recognizes tau phosphorylated at threonine residues 212 and 217 22 . Phosphorylation of amino acid 217 is the minimally required epitope, yet phosphorylation of amino acid 212 enhances binding, and thus the recognition is termed “p217+tau.” As described for the CSF p217+tau assay, measurement of species containing both p212 and p217 may yield additional diagnostic relevance than measurement of just one of these residues 19 . Indeed, while liquid chromatography/mass spectrometry (LC/MS) studies of paired helical filament (PHF) tau revealed monophosphorylated peptides containing pT217 alone, pT212 was only found in peptides that also contained phosphorylation of S214 or T217 14 .…”

“…Predominantly observed species are comprised of N‐terminal, mid‐region, and select microtubule binding region (MTBR) epitopes, and additional major cleavage sites are likely in the region from aa 70‐120 and at ~aa 224 15,26,34,35,36 . Fractionation of CSF prior to assay has shown that CSF p217+tau is also entirely of fragmented nature, lacking C‐terminus and potentially also cleaved between aa7‐119 19 . Here we show, by comparing the p217+tau “short” and p217+tau “long” assay results, that plasma p217+tau may present a similar level of fragmentation in the aa 7‐119 region as in CSF.…”

Plasma p217+tau vs NAV4694 amyloid and MK6240 tau PET across the Alzheimer continuum

“…More recently a high sensitivity Simoa assay has been developed using a capture antibody (pT3) that was raised against tau in paired helical filaments (PHF) of AD brain [12,13]. The core requirement for this antibody binding is phosphorylation at aa217 (p217) with enhanced binding when other nearby phosphorylated sites are present, predominantly at aa212 [12].…”

CSF p-tau increase in response to Aβ-type and Danish-type cerebral amyloidosis and in the absence of neurofibrillary tangles

“…We then used an immunoassay which allows the quantification of tau phosphorylated at threonine 217 with or without adjacent phospho-epitopes (“p217 + tau” [ 12 ] (see also Supplementary Methods, online resource). P217 + tau also showed an age-dependent increase and reached a plateau, although at 14- to 16-fold higher levels compared to 1.5-month-old APPPS1 tg animals (Fig.…”