Simoa ptau 231 advantage kit

M-PER^® Mammalian Protein Extraction Reagent (#78503 Pierce) was added to the isolated EV fraction using MagCapture Exosome Isolation Kit PS with Halt™ Protease and Phosphatase Inhibitor Cocktail (#78442 Thermo Fisher Scientific) and was mixed by vortexing for 15 min. The lysed EVs were filtered by 0.45 μm Spin-X centrifuge tube (#CLS8162 Corning). The EV t-tau and p-tau231 were measured using the Simoa Tau advantage kit (#101522 Quanterix, Lexington, KY, USA) and Simoa pTau-231 Advantage kit (#102292 Quanterix) on the Simoa HD-1 analyzer (Quanterix). All CSF-derived EV samples were diluted 10x with the Tau Calibrator Diluent (#101631 Quanterix) prior to the assays, to minimize matrix effects, and were analyzed in duplicate on one occasion. The relative concentration estimates of t-tau and p-tau231 were calculated according to the standard curve.

To collect CSF samples from the monkeys, a lumbar puncture was performed with a 22 G traumatic needle between L4 and L5. The CSF samples were transferred to sterile low-binding microcentrifuge tubes and then centrifuged at 10,000 g at 4 °C for 5 min. Next, the total tau, phospho-tau 181, phospho-tau 231, Aβ42, Aβ40, and NfL peptide levels were analysed for all samples using Quanterix’ ultrasensitivity digital biomarker detection technology with the Simoa® HD-X Analyser™ (Quanterix, Billerica, MA, USA), the latest model of the fully automated Simoa bead-based immunoassay platform. The assay kits used in this study were the Simoa® Neurology 3-Plex A Kit (101,995, Quanterix, USA), the Simoa™ pTau-181 V2 Advantage Kit (103,714, Quanterix, USA), the Simoa™ pTau-231 Advantage Kit (102,292, Quanterix, USA), and the Simoa™ NF-light V2 Advantage kit (104,073, Quanterix, USA).

A Simoa HD-X analyzer (Quanterix, Lexington, MA) was used to measure phosphorylated tau (p-Tau) 181 (SIMOA pTau-181 Advantage V2 kit, Quanterix, Lexington, MA), Aβ40, Aβ42, glial fibrillary acidic protein (GFAP), and neurofilament light (NFL) (SIMOA Neuro 4-Plex E Advantage kit, Quanterix, Lexington, MA) concentrations in plasma and CSF. The SIMOA pTau-231 Advantage kit (Quanterix, Lexington, MA) was used to measure pTau 231 concentration in CSF. Prior to loading the samples onto the Simoa analyzer, plasma and CSF samples were clarified by centrifugation at 14,000 x g for 10 minutes. All samples were run in duplicate. In addition, a Fujirebio G1200 (Malvern, PA) was used to measure total tau (lumipulse G total tau, Malvern, PA), pTau-181 (lumipulse G pTau-181, Malvern, PA) Aβ40 (lumipulse G B-Amyloid 1–40, Malvern, PA), and Aβ42 (lumipulse G B-Amyloid 1–42, Malvern, PA) as per the manufacturer’s protocol.