Simoa hd 1 platform

For sample types in which the levels of FL tau were too low to measure by ELISA (i.e., CSF and plasma exosome lysates), tau was measured using a Simoa (single molecule analysis)-based version of our FL assay and a Simoa HD-1 platform (Quanterix, Lexington, MA, USA). The C-terminal antibody, TauAB (a generous gift from Andy Billinton and Mike Perkinton of MedImmune), was used for capture and the N-terminal Tau12-biotin antibody (Cat. #MAB2241; Millipore, Billerica, MA, USA) for detection. Beads were activated with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Cat. #22980; ThermoFisher, Waltham, MA, USA) and coated with TauAB. Tau12 was biotinylated using N-hydroxysuccinimide-polyethylene glycol (NHS-PEG4)-biotin (Cat. #21330; ThermoFisher, Waltham, MA, USA). Standards (human tau 381; Cat. #T0201; MilliporeSigma, St. Louis, MO, USA) and blanks were prepared using sample buffer and assayed in triplicates while samples were assayed in duplicate. Standard curves were fitted and LLoQs determined as with the mid-region and p181 tau assays. For the experiments shown, the LLoQ of the FL Simoa assay was 0.74 pg/mL. Full validation of this assay will be described in a separate publication [79 ].

Venous blood was collected in tubes containing EDTA and centrifuged at 2,000 × g for 10 min at 4°C. The obtained plasma was divided into ~500 μl aliquots and frozen at −80°C. All samples underwent no more than three freeze-thaw cycles (Keshavan et al., 2018 (link)). The samples were rapidly thawed at 22 ± 2°C and then centrifuged at 10,000 × g prior to analysis to prevent any sample debris from interfering with the measurements. Plasma Aβ42, Aβ40, t-tau, and NfL concentrations were measured simultaneously using the single-molecule array (SIMOA)-HD1 platform (SIMOA; Quanterix, Billerica, MA, USA), which employed an automated SIMOA principle. Briefly, Aβ42, Aβ40, and t-tau levels were measured using a multiplex array (Neurology 3-Plex A Advantage Kit, N3PA), and NfL levels were measured using a single-analyte array (NF-light). The samples were measured using a two-step immunoassay. All analytical procedures were performed according to the protocol of the manufacturer by well-trained technicians who were blinded to the state of participant and clinical data, according to the protocol of the manufacturer. Samples with coefficients of variance (CV) of >20% were excluded from the analyses. In this study, the within-batch CV of all samples was <5%.

Serum cytokines IL-6, IL-10, TNFα and IL-1β were quantified using the Simoa HD-1 platform from Quanterix (Billerica, MA) according to manufacturing guidelines and as specified by Stukas et. al (55 (link)).