B-051 Automation of the VeraBIND Tau Plasma Assay to Detect the Seeding Activity of Hyperphosphorylated Tau Before the Onset of Symptomatic Alzheimer’s Disease

Abstract

Alzheimer’s disease is characterized by neurodegeneration of the brain associated with beta amyloid and tau pathologies. In tau pathology, hyperphosphorylated tau (pTau) binds to normal tau in a prion-like manner forming insoluble tau aggregates. Here, we evaluate the performance characteristics of the recently automated VeraBIND® Tau assay which isolates pTau from plasma and tests its functional ability to bind recombinant normal tau, moving beyond “concentration-based” biomarker detection to “activity-based” biomarker detection of pathologically active hyperphosphorylated tau (PA pTau). The VeraBIND® Tau assay uses a pool of different antibody-coated capture beads to capture pTau in the plasma sample (Anti-pTau Solid Phase). The beads are washed to remove the plasma matrix, and buffer-exchanged/washed into a normal tau binding buffer that mimics the pH, ionic and hydrophobic binding environment in the brain. The assay adds recombinant full-length normal tau 1-441 with a recombinant tag at the C-terminus (Recombinant Normal Tau-441) to determine if any of the pTau captured on the beads is PA pTau. If the pTau captured from the plasma is PA pTau, it will bind Recombinant Normal Tau-441 like it does in the brain with tau pathology. The final step washes the capture beads to remove unbound Recombinant Normal Tau-441, adds an alkaline phosphatase conjugated anti-tag monoclonal antibody (Anti-Tag AP Conjugate), washes the capture beads to remove unbound Conjugate, and adds Substrate to generate a relative luminescence signal (RLU) that is directly proportional to the amount of Recombinant Normal Tau-441 bound by the PA pTau in the plasma sample. The VeraBIND Tau assay protocol was recently automated using a Tecan Fluent® 1080 liquid handler with accessories, equipment and consumables including a flexible channel arm, multi-channel arm, robotic gripper arm, Incubated Hotel, 5-position Hotel for deep well plates, Alpaqua Magnum FLX® (2. Generation) plate magnets, Qinstrument BioShake Q1 heater-cooler shakers with thermal adapters, Promega GloMax® Discover Microplate Reader, 2 mL deep well plates, and white round bottom luminescence read plates. The fully automated Tecan assay protocol has a throughput of 360 tests per day with a time to result of 8 hours and 40 min. When running a full-plate assay, the Positive Control demonstrates duplicate CVs of < 2% and the Negative Control and Standard demonstrate duplicate CVs <15%. When 2 different negative EDTA plasma samples collected from healthy controls were tested they demonstrated overall CVs of 5.8% (N=12) and 9.9% (N=74). The accuracy of the automated Tecan assay protocol to detect PA pTau in plasma will be validated using a large validation cohort of well characterized plasma collected from tau PET positive (T+) and tau PET negative (T-) individuals, and results compared to the semi-automated assay protocol (N=145; overall accuracy 96.7% to distinguish T+ from T- individuals with 94.2% sensitivity, 96.1% specificity and AUC 0.974). The automated VeraBIND Tau plasma assay provides the throughput, precision and accuracy required to detect PA pTau in cognitively unimpaired individuals not only for confirming disease pathology, but also for screening, trial recruitment, early intervention, and monitoring drug efficacy.