Targeting multiphosphorylated tau: technical and clinical validation of a new Simoa® assay for CSF and plasma detection of tau simultaneously phosphorylated at T181 and T231

Abstract

Background and Objectives: Different forms of phosphorylated tau (p-tau) have shown high potential as Alzheimer′s Disease (AD) biomarkers in both cerebrospinal fluid (CSF) and plasma. Hence, we hypothesized that tau peptides showing concomitant phosphorylation at two different sites may provide an increased diagnostic value. We therefore developed and validated a new Simoa® immunoassay detecting tau simultaneously phosphorylated at T181 and T231 (C231D181) in cerebrospinal fluid (CSF) and plasma. Methods: Technical validation of the C231D181 Simoa® assay included standard curve development, assessment of antibodies cross-reactivity, dilutional linearity, sensitivity, as well as intra- and inter-assay precision. Subsequently, we measured CSF C231D181, p-tau181, and p-tau231 in two cohorts: discovery (MCI-AD n=21, AD dementia n=19, CTRL n=15) and validation (preclinical AD n=19, MCI-AD n=20, AD dementia n=16, frontotemporal dementia n=39, CTRL n=24). Additionally, in the discovery cohort, C231D181, p-tau181, and p-tau231 levels were measured in matched plasma samples. Results: Specificity of the assay was assessed using a synthetic peptide simultaneously phosphorylated at T181 and T231, while cross-reactivity was excluded with a mix of single-site phosphorylated peptides (T181 or T231). Both in discovery and validation cohorts, CSF C231D181, p-tau181, and p-tau231 levels were significantly elevated in all AD groups vs. CTRL. As assessed in discovery cohort, plasma p-tau231 and p-tau181 levels enabled effective discrimination of AD continuum groups from CTRL (AUC plasma p-tau231: CTRL vs. MCI-AD=0.925, CTRL vs. AD-dem=0.947; AUC plasma p-tau181: CTRL vs. MCI-AD=0.877, CTRL vs. AD-dem=0.943) while plasma C231D181 did not change among clinical groups. Discussion: A new ultrasensitive immunoassay detecting tau simultaneously phosphorylated at T181 and T231 was developed and validated. While we found this phosphorylated tau form to be significantly elevated across the AD continuum in CSF, in plasma it did not show changes among the diagnostic groups. The differences between CSF and plasma suggest matrix-specific protein processing. Our findings support evidence for qualitative and quantitative importance of tau phosphorylation across AD continuum and warrant further investigation, including assessment of tau simultaneously phosphorylated at multiple sites.