B-260 A Novel Bioluminescent, Multi-Epitope Assay to Detect Alzheimer’s Disease-Specific Tau Consistent with Alzheimer’s Amyloidosis

Background Early and accurate detection of amyloidosis and diagnosis of Alzheimer’s disease (AD) is crucial for effective management and treatment with new FDA-approved amyloid beta plaque-reducing therapeutics such as Lecanemab and Donanemab. Amyloid positron emission tomography (PET) imaging is the gold standard for detecting amyloid beta plaque in the brain but is expensive, requires radioactive tracers, and presents significant logistical challenges. Cerebrospinal fluid (CSF) biomarker testing is invasive and poses risks to patients. Recently, blood-based biomarkers, particularly phosphorylated Tau (pTau) assays, show promise in detecting amyloidosis and aiding AD diagnostics in patients with cognitive decline. However, existing pTau assays exhibit variability in sensitivity and specificity, leading to a diagnostic "grey zone" in 10–30% of tested patients. Given these limitations, there is a significant unmet need for a simple, non-invasive blood test that accurately detects amyloid pathology for early detection and disease progression monitoring. Here, we developed an ultra-sensitive blood test that detects Alzheimer’s disease-specific tau (AD Tau), enabling both early and late-stage amyloid beta plaque detection. Methods A novel multi-epitope assay was designed to capture, purify, and detect AD Tau. EDTA plasma from amyloid PET-confirmed individuals (amyloid beta plaque absent, n=23; amyloid beta plaque present, n=27) was pre-analytically conditioned and cleared of heterophilic and autoantibody interferences using clean beads. AD Tau was captured and purified using capture beads coated with multi-epitope targeting antibodies. Following biomarker capture, AD Tau peptides were eluted and neutralized into assay buffer containing non-ionic detergent to maintain peptide solubility. A ternary split-NanoLuc luciferase complementation reporter system was used as the detection module. This system used two small reporter peptides appended to AD Tau-specific antibody reagents and a polypeptide protein. Binding of the tagged antibodies to two different AD Tau-specific epitopes on purified AD Tau generates a stable bioluminescent signal through proximity-induced complementation between the reporter peptides and polypeptide protein in the presence of a luminogenic substrate. The AD Tau biomarker was detected using a luminescent plate reader. A calibrator was used to derive a ratio-metric value from the assay output, which facilitated simple yes/no results based on a predefined cutoff value of 1.000. Results Semi-quantitative analysis of 50 plasma samples showed that individuals with PET-confirmed amyloid beta plaque deposits had significantly higher AD Tau levels than those without (p < 0.0001). Categorizing the assay results into a binary ‘Yes/No’ outcome demonstrated an Overall Percent Agreement (OPA) of 88.0% and an Area Under the Receiver Operating Characteristic Curve (AUC-ROC) of 93.0%, indicating high reliability and accuracy compared to amyloid PET imaging. Conclusions This study successfully demonstrated a highly sensitive and non-invasive multi-epitope assay for detecting AD Tau, which is indicative of amyloidosis, potentially marking a significant advancement in AD detection. Using sample pre-conditioning, sample cleaning, and biomarker purification, paired with a bioluminescent, homogeneous analyte detection system, this non-invasive assay can be used to facilitate early detection and enhance accuracy for amyloidosis and AD. This innovative approach has the potential to revolutionize the management and treatment of AD, enabling earlier therapeutic interventions and improved patient outcomes.