Non-Phosphorylated Tau as a Potential Biomarker of Alzheimer's Disease: Analytical and Diagnostic Characterization

Background: Virtually nothing is known about a potential diagnostic role of non-phospho-epitopes of Tau (Non-P-Tau) in cerebrospinal fluid (CSF). Objective: To establish and analytically and clinically characterize the first assay capable to measure concentrations of Non-P-Tau in human CSF. Methods: An antibody (1G2) was developed that selectively binds to the Tau molecule non-phosphorylated at the positions T175 and T181, and was used in establishing a sandwich ELISA capable to measure Non-P-Tau in human CSF, following analytical and clinical validation of the method. Results: The 1G2 antibody shows decreasing reactivity to tau peptides containing phosphorylation mainly at positions T175 and T181. Detection limit of the assay is 25 pg/ml; the coefficients of variation (CVs) of the optical densities of the repeated standard curves were between 3.6-15.9%. Median intra-assay imprecision of double measurements was 4.8%; inter-assay imprecision was in the range of 11.2%-15.3%. Non-P-Tau concentrations are stable in the CSF samples sent to distinct laboratories under ambient temperature; inter-laboratory variation was approximately 30%. The Non-P-Tau CSF concentrations were highly significantly increased in patients with Alzheimer's disease in stage of mild cognitive impairment or dementia (AD/MCI, n = 58, 109.2 +/- 32.0 pg/mL) compared to the non-demented Controls (n = 42, 62.1 +/- 9.3 pg/mL, p < 0.001). At the cut-off of 78.3 pg/mL, the sensitivity and the specificity were 94.8% and 97.6%, respectively. Conclusion: For the first time, an assay is reported to reliably measure concentrations of non-phosphorylated Tau in human CSF.