Blood samples were collected and processed according to the ADNI protocol (Kang et al., 2015 (link)). Plasma p-tau181 concentrations were measured at the Clinical Neurochemistry Laboratory, University of Gothenburg (Mölndal, Sweden) using an assay developed in-house on a Simoa HD-X (Quanterix) instrument, as described previously in detail (Karikari et al., 2020 (link)). In brief, the AT270 mouse monoclonal antibody (MN1050; Invitrogen) specific for the threonine-181 phosphorylation site, coupled to paramagnetic beads (103 207; Quanterix) was used for capture and the anti-tau mouse monoclonal antibody Tau12 (806 502; BioLegend), which binds the N-terminal epitope 6-QEFEVMEDHAGT-18 on human tau protein, for detection. All of the available samples were analysed in a single batch. We identified four participants (0.4%) with outlier values of plasma p-tau181 levels that were discarded from subsequent analyses (Supplementary Fig. 1 ). Longitudinal blood sampling was performed approximately every year, over a median follow-up time of 2.9 years in 938 subjects.
Simoa hd x
Manufactured by Quanterix
Sourced in United States
The Simoa HD-X is a high-definition digital immunoassay analyzer developed by Quanterix. It is designed to detect and quantify low-abundance proteins and other analytes with high sensitivity and specificity. The Simoa HD-X uses Quanterix's proprietary Single Molecule Array (Simoa) technology to enable the detection of individual protein molecules, providing a powerful tool for researchers and clinicians.
Automatically generated - may contain errors
Lab products found in correlation
Sig 39416, by BioLegend (2 mentions)
Tau 2.0 sample diluent, by Quanterix (2 mentions)
Simoa neurology 4 plex e, by Quanterix (2 mentions)
Tau12, by BioLegend (1 mentions)
Mn1050, by Thermo Fisher Scientific (1 mentions)
Paramagnetic beads, by Quanterix (1 mentions)
Simoa nfl advantage kit, by Quanterix (1 mentions)
Simoa nf light advantage kit, by Quanterix (1 mentions)
Simoa neurology 4 plex e advantage kit, by Quanterix (1 mentions)
Nf light kit, by Quanterix (1 mentions)
27 protocols using simoa hd x
1
Plasma p-tau181 Measurement Protocol
2
Serum Biomarker Measurement Protocol
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Serum samples were obtained at the indicated time points according to standard procedures and stored at −80 °C until use. Serum BD-tau and p-tau231 were measured on the Simoa HD-X platform (Quanterix) using validated in-house assays,11 (link),14 (link) and T-tau and NfL with Quanterix assays (Nos. 101552 and 103670, respectively).
3
Quantitative CSF p-tau235 Measurements
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CSF p-tau235 measurements were performed at the Clinical Neurochemistry Laboratory at the University of Gothenburg using a Simoa HD-X instrument (Quanterix), and all samples were blinded. Prior to the analysis, samples were allowed to thaw at room temperature for 45 min. Thawed samples were subsequently vortexed for 15 s, after which they were plated and diluted 1:2 using Tau2.0 sample assay diluent (Quanterix). Due to sample volume availability, CSF samples from the Paris cohort were run in singlicates, while BIODEGMAR cohort samples were run in duplicates. An eight-point calibration curve was generated using recombinant full-length GSK-3β-phosphorylated tau-441 (SignalChem) and run in duplicates. Two internal quality controls (iQC, low and high) were run at the beginning and end of each plate, also in duplicates. Repeatability (%CVr) and intermediate precision (%CVRw) values for both cohorts were ˂15%. Further details on assay specifications and validation can be found elsewhere [15 (link)].
4
SARS-CoV-2 Spike IgG Antibody Quantification
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SARS-CoV-2 Spike IgG antibodies were quantified automatically in patient sera by a Simoa immunoassay using the Simoa HD-X analyser (Quanterix, Billerica, MA, USA). Samples were analyzed using the commercial SARS-CoV-2 Spike IgG Advantage kit (item 103769), using a similar procedure as for the SARS-CoV-2 N antigen. The positivity cut-off of 924 ng/mL corresponding to the maximal value obtained in pre-pandemic serum samples was used [24 ]. SARS-CoV-2 antibodies were available for 233 samples from 84 patients out of the 90 (93.3%) due to insufficient residual serum samples.
5
Plasma p-tau212 Quantification Protocol
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For the p-tau212 assay, which was developed on the Simoa HD-X instrument (Quanterix, MA, USA), the p-tau212 antibody was used as the capture antibody. A mouse monoclonal antibody raised against the N-terminal region of tau (Tau12; BioLegend, #SIG-39416) was used for detection. In vitro phosphorylated recombinant full-length tau-441 (#269022, Abcam) was used as the assay calibrator. Blood samples and calibrators were diluted with assay diluent (Tau 2.0 Sample Diluent; #101556, Quanterix).
Analytical validation of the p-tau212 assay followed protocols described previously8 (link),10 (link),31 ,39 (link). Assay development work was conducted at the University of Gothenburg, Sweden. In the clinical studies, p-tau212 was measured at the University of Gothenburg by scientists blinded to participant information, using the above-described assays. For p-tau212, plasma and CSF samples were diluted 1.2 times and 10 times respectively prior to measurement.
Analytical validation of the p-tau212 assay followed protocols described previously8 (link),10 (link),31 ,39 (link). Assay development work was conducted at the University of Gothenburg, Sweden. In the clinical studies, p-tau212 was measured at the University of Gothenburg by scientists blinded to participant information, using the above-described assays. For p-tau212, plasma and CSF samples were diluted 1.2 times and 10 times respectively prior to measurement.
6
Plasma Biomarkers and Neuroimaging
7
Plasma Biomarker Assessment for Alzheimer's
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Phlebotomy was performed at the KU Clinical and Translational Science Unit following an overnight fast. Whole blood was stored for APOE genotyping and further processed to generate plasma as previously described [11 (link)]. For this study, pTau181 was measured in plasma on a Simoa HD-X (Quanterix, Billerica, MA) according to the manufacturer’s instructions. Data on amyloid-β 42 (Aβ42), amyloid-β 40 (Aβ40), neurofilament light (NFL), and glial fibrillary acidic protein (GFAP) measured previously on the Simoa HD-X using the Neurology 4-Plex E kit were also statistically analyzed in relation to our new outcomes of interest [11 (link)].
8
Plasma and CSF p-tau212 Assay Protocol
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For the p-tau212 assay, which was developed on the Simoa HD-X instrument (Quanterix, MA, USA), the p-tau212 antibody was used as the capture antibody. A mouse monoclonal antibody raised against the N-terminal region of tau (Tau12; BioLegend, #SIG-39416) was used for detection. In vitro phosphorylated recombinant full-length tau-441 (#269022, Abcam) was used as the assay calibrator. Blood samples and calibrators were diluted with assay diluent (Tau 2.0 Sample Diluent; #101556, Quanterix).
Analytical validation of the p-tau212 assay followed protocols described previously8 (link),10 (link),31 ,39 . Assay development work was conducted at the University of Gothenburg, Sweden. In the clinical studies, p-tau212 was measured at the University of Gothenburg by scientists blinded to participant information, using the above-described assays. For p-tau212, plasma and CSF samples were diluted 1.2 times and 10 times respectively prior to measurement.
Analytical validation of the p-tau212 assay followed protocols described previously8 (link),10 (link),31 ,39 . Assay development work was conducted at the University of Gothenburg, Sweden. In the clinical studies, p-tau212 was measured at the University of Gothenburg by scientists blinded to participant information, using the above-described assays. For p-tau212, plasma and CSF samples were diluted 1.2 times and 10 times respectively prior to measurement.
9
Plasma Collection and Quantification
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Blood was collected at each clinic visit by venous puncture using 10‐mL K2 EDTA tubes. The tube was immediately inverted 5 to 10 times, then was spun for 10 min at 2000 × g and plasma was collected in 1‐mL aliquots and stored at −80°C. When ready for quantification, plasma was thawed on ice and centrifuged at 4°C for 10 min at 21,000 × g. Samples were run on the Quanterix Simoa HD‐X platform in duplicate using the same settings as previously described (Table S1 ).
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10
Plasma Neurofilament Light Quantification
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Blood was collected in BD EDTA tubes and then centrifuged at 2500 × g for 10 min at 4 °C to obtain plasma for each participant. Plasma samples were diluted at a ratio of 1:4. The single-molecule (Simoa) array technology by an ultra-high sensitivity protein molecular detection instrument (Simoa HD-X, Quanterix, MA, USA) and the Simoa NfL Advantage kit (Quanterix, MA, USA) measured the plasma NfL level [41 (link)]. All NfL values were within the linear ranges of the assays. The average intraassay coefficient of variation was 4.94%.
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