Validation of ELISA Methods for Quantification of Total Tau and Phosphorylated-Tau 181 in Human Cerebrospinal Fluid with Measurement in Specimens from Two Alzheimer's Disease Studies

Tau measurements in cerebrospinal fluid (CSF) are gaining acceptance as aids to diagnosis of Alzheimer's disease (AD) and differentiation from other dementias. Two ELISA assays, the INNOTEST^® hTAU Ag and the INNOTEST^® PHOSPHO-TAU_(181P) for quantification of t-tau and p-tau₁₈₁ respectively, have been validated to regulatory standards. Validation parameters were determined by repeated testing of human CSF pools. Specimens from Phase 2 studies of the γ-secretase inhibitor semagacestat and the therapeutic antibody solanezumab at baseline and following 12–14 weeks of treatment were also tested. Estimated intra-assay CV for repeated testing of 3 CSF pools were ≤11.5% and RE varied between −14.1% and +6.4%. Inter-assay CV for t-tau was <5% and RE was within ±8%. For p-tau₁₈₁, inter-assay CV was <9% and RE was within ±2.5%. Total CV (intra-assay plus inter-assay) were below 10% for both analytes. Up to 20-fold dilutional linearity was demonstrated for both analytes. Stability of t-tau and p-tau₁₈₁ was demonstrated in CSF during five freeze-thaw cycles at ≤−20°C and ≤−70°C and at 18–22°C for up to 24 h. Neither semagacestat nor solanezumab interfered with either assay. Inter-individual t-tau and p-tau₁₈₁ concentrations were highly variable but intra-individual variations were small. These assays are suitable for analysis of CSF t-tau and p-tau₁₈₁ in a single laboratory supporting multi-center AD clinical trials. No effect of treatment with semagacestat or solanezumab was observed in response to three months of drug administration.