B-261 Performance Validation of a plasma-based Immunoassay for P-tau217

Background Phosphorylated tau protein has shown significant diagnostic accuracy and specificity leading to earlier identification of Alzheimer’s disease (AD). Specifically, plasma P-tau217 has demonstrated accuracy comparable to other tau cerebrospinal fluid (CSF) and blood-based biomarkers in determining AD pathology. The Quanterix SP-X Human P-tau217 immunoassay (Quanterix, Corp., Billerica, MA) is a chemiluminescent sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative measurement of P-tau217 in human EDTA plasma. The ELISA incorporates antibodies developed by Eli Lilly and Company (Indianapolis, IN) that include a brain P-tau217 specific antibody for capture, and a secondary antibody specific for brain tau. The intensity of the luminescent signal produced is directly proportional to the concentration of P-tau217 in the sample. Results are expressed as U/mL. Methods Specimens were collected under University of Utah’s Institutional Review Board approved protocols or provided by Eli Lilly and Company. Samples consisted of elevated tau CSF spiked plasma and plasma from a cohort of patients presenting with cognitive decline. Measurements were conducted according to the kit manufacturer’s protocol. Two SP-X imagers were validated. Performance characteristics evaluated included a method comparison against the CertuitAD® P-tau217 assay (Eli Lilly Clinical Diagnostics Laboratory), analyte recovery, precision, analytical sensitivity, analyte stability, and interference/cross-reactivity. Reference cut-points for classifying disease status were also established. Results A method comparison study against CertuitAD® assay generated a slope of 0.965, intercept of 0.021, r2 of 0.928 and bias of 6.0% (n=396, Deming regression, Bland-Altman analysis). Recovery was assessed by combining calibration materials at different ratios, creating six specimens of varying P-tau217 concentrations. Percent recoveries ranged 87.3–114.8%. Precision was determined for two imagers and two kit lots utilizing three CSF spiked plasma pools of differing P-tau217 concentrations tested over six days, four replicates per pool per day. Repeatability CVs ranged 3.3–6.3, 3.5–5.8 and 5.1–9.6% for High, Mid and Low P-tau217 concentrations, respectively. Within-laboratory CVs were 4.4–8.8, 4.8–6.9 and 9.3–12.4% for High, Mid and Low concentrations, respectively. The limit of detection was 0.04 U/mL (26 determinations each of blank material and a low-level plasma pool). The limit of quantitation was established at 0.07 U/mL. P-Tau217 protein stabilities were determined for minimums of 48 hours, 7 days, 2 weeks and 3 cycles for ambient, refrigerated, frozen (-20 °C) and freeze/thaw, respectively (±20% from baseline). Interference (outside ±20% from matrix-matched controls) was evident above 300 and 4.3 mg/dL for hemoglobin and bilirubin, respectively. Lipemia and rheumatoid factor did not interfere up to 1500 mg/dL and 99 IU/mL, respectively. Drug interference results, involving a total of 47 drug compounds, and cross-reactivity with P-tau181, were also within the above stated acceptable limits. Analysis of positron emission tomography combined with P-tau217 results from individuals presenting with cognitive decline (n=524, ages 60–86 years), established cut-offs of Negative, <0.13 U/mL; Indeterminate, =0.13 to <0.20 U/mL; and Positive, =0.20 U/mL. Conclusion The Quanterix SP-X Human P-tau217 immunoassay demonstrates acceptable performance for quantifying P-tau217 in human plasma. Results support the assay as an aid in the detection of AD pathology.