AD pathology (Aβ40, Aβ42, phosphorylated tau at threonine 181 [p-tau181]) and neurodegeneration (neurofilament light [NfL]) were measured using commercially available ultrasensitive single-molecule array (Simoa) assays from Quanterix using stored plasma (visits 3, 5, and 6/7).17 (link) Aβ40, Aβ42, and NfL were measured using the Neurology 4-PLEX E. Glial fibrillary acidic protein was excluded from the study because of our specific focus on AD-related neuropathology and neurodegeneration. Aβ42:40 ratio was calculated by dividing Aβ42 over Aβ40. Biomarkers of p-tau181 and NfL were right skewed and base 2 log transformed. Lower plasma Aβ42:40 levels and higher p-tau181 levels reflect greater Aβ pathology in early stages of AD development, and higher and faster increasing levels of NfL reflect greater neurodegeneration.18 (link)
Simoa assay
Manufactured by Quanterix
Sourced in United States
The Simoa assay is a highly sensitive and precise technology developed by Quanterix for the detection and quantification of proteins and other biomolecules. It utilizes digital enzyme-linked immunosorbent assay (ELISA) technology to enable the measurement of low-abundance analytes with exceptional accuracy and reproducibility.
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Lab products found in correlation
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Tau12, by BioLegend (1 mentions)
Simoa nf light assay, by Quanterix (1 mentions)
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Bovine nfl, by UmanDiagnostics (1 mentions)
Neurology 3 plex a advantage kit, by Quanterix (1 mentions)
17 protocols using simoa assay
1
Plasma Biomarkers for Alzheimer's Disease
2
Quantitative Plasma Biomarker Assays
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Plasma was collected by venipuncture, processed, and stored in aliquots at − 80 °C according to the standardised procedures. Plasma NfL concentration was measured using a commercial single-molecule array (Simoa) NF-Light® assay (Quanterix, Billerica, MA) according to the manufacturer’s instructions [25 ]. Plasma p-Tau181 concentration was measured using an in-house Simoa assay developed at the University of Gothenburg [8 (link)]. In brief, the capture antibody (AT270, Invitrogen) which is specific for the threonine-181 phosphorylation site [26 (link)] was coupled to paramagnetic beads whilst the detector antibody (Tau12, BioLegend) was raised against the N-terminal epitope amino acid 6-QEFEVMEDHAGT-18 on human tau protein. Detailed analytical procedures and assay validation have been previously described [8 (link)]. Plasma GFAP, Aβ1–42, and Aβ1–40 concentrations were measured using commercial Simoa assays (Quanterix, Billerica, MA). All measurements were carried out using an HD-X analyser (Quanterix, Boston, MA) in one round of experiments, using one batch of reagents with operators blinded to clinical information.
3
Serum Biomarkers Measurement Protocol
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Serum concentration of sNfL was measured using an in house single molecule array (Simoa) method on an HD-1 analyzer (Quanterix, Lexington, MA), as previously described in detail by Gisslen [22 (link), 23 (link)]. Serum concentrations of tau and GFAp were measured using commercially available Simoa assays (Quanterix, Lexington, MA). All samples were measured as duplicates. The mean coefficient of variation of duplicate concentrations was 4.3%. In addition, a quality control sample was measured in duplicate on each of the seven runs used to complete the study. The intra-assay coefficient of variation for this sample was < 10%. All measurements were performed by board-certified laboratory technicians. The laboratorial analysis was conducted in a blinded fashion, as the assays were done without knowledge of the patient’s identity or diagnosis. Only one round of experiments was performed, using one batch of reagents.
4
Serum NfL and TNF-α Quantification
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Mice were anesthetized and blood samples were collected. The collected blood was incubated at room temperature for 30 min to enable clot formation and centrifuged at 1500 g for 10 min. The serum was frozen and stored at −80 °C until analysis. For NfL measurement, sera were initially diluted at 1:1000. When an obtained concentration was higher than 500 pg/mL, an additional dilution of 1:5000 was further tested. For tumor necrosis factor α (TNF-α), sera samples were diluted 1:8. NfL concentrations were measured in duplicates by a single molecule array (Simoa) assay (Quanterix, Boston, MA, USA) employing commercial kits (NF-light Advantage kit and mouse TNF-α discovery kit for HD-1/HD-X adjusted for SR-X, UmanDiagnostics Umea, Sweden), using a bead-conjugated immunocomplex. The immunocomplex was applied to a multi-well array designed to enable imaging of every single bead. The average number of enzymes per bead (AEB) of each sample was interpolated onto the calibrator curve constructed by AEB measurements on commercial NfL and TNF-α (UmanDiagnostics), serially diluted in an assay diluent. Samples were analyzed using one batch of reagents. Animal treatment information was blinded for the investigator performing the analysis.
5
Quantification of Neurofilament Light
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CSF and serum NfL levels were determined using the Simoa® assay (Quanterix). Assay methodology has been previously described [28 (link)]. Laboratory personnel were blinded to experimental groups and time points.
6
Quantitative IFNα Detection Assay
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As described by Rodero et al., the Simoa IFNα assay was developed using a Quanterix Homebrew Simoa assay according to the manufacturer’s instructions and utilising two autoantibodies specific for IFNα isolated and cloned from 2 APS1/APECED patients [9 (link), 12 (link)]. The 8H1 antibody clone was used as a capture antibody after coating on paramagnetic beads (0.3 mg/mL), and the 12H5 was biotinylated (biotin/Ab ratio = 30/1) and used as the detector. Recombinant IFNα17/αI (PBL Assay Science) was used as a standard curve after cross-reactivity testing. The limits of detection (LOD) were calculated by the mean value of all blank runs + 3SDs and was 0.23 fg/ml.
7
Quantification of Serum/Plasma NfL
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Frozen serum and plasma samples were thawed on ice and were used immediately; repeated freezing and thawing of the samples was avoided. NfL concentrations in samples were measured using the Simoa™ assay (Catalog # 103186; Quanterix, Billerica, MA, USA). Samples were diluted 1:4 and randomly distributed on 96‐well plates. Quality control (QC) samples provided with the kit had concentrations within the pre‐defined range and the coefficient of variance (CV) across the plates was <10%: (1) cohort 1: 1 plate, no CV; (2) cohort 2: 3 plates, control 1 = 8.9% and control 2 = 3.7%; (3) cohort 3: 5 plates, control 1 = 8.6% and control 2 = 7.6%. All samples were analyzed blindly under alpha‐numeric codes. The diagnostic codes were broken only after QC‐verified NfL concentrations were reported to the database manager.
To determine the effect of sample age on NfL degradation and assay performance, within each cohort and disease diagnosis/severity subgroups, we analyzed the correlations between sample age (date of sample analysis – date of sample collection, in days;Data S1 ) and NfL concentrations (pg/mL) using linear regression models. We did not observe statistically significant (p < 0.05) correlations (data not shown).
To determine the effect of sample age on NfL degradation and assay performance, within each cohort and disease diagnosis/severity subgroups, we analyzed the correlations between sample age (date of sample analysis – date of sample collection, in days;
8
Serum NF-L Quantification by Simoa
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Serum samples were thawed, and the concentration of neurofilament light chain (NF-L) was measured with a single-molecule array (Simoa) assay (Quanterix, Billerica, MA) according to the manufacturer's protocol.
9
Ultrasensitive IFNα Quantification Assay
10
Serum Neurofilament Light Chain Longitudinal Assessment
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Serum samples were collected annually as part of the CLIMB study at the time of patient visits and stored at −80°C. Using these collected samples, the sNfL analysis was performed retrospectively using single‐molecule array (SIMOA) assay (Quanterix Corp, Boston, MA, US) for the purposes of this study. The date of first sNfL assessment (at enrollment) was defined as the index date (baseline). Serum was also obtained at year one (approximately 12 months from baseline) and at year two (approximately 24 months from baseline) and sNfL was analyzed retrospectively for this study. Log‐transformed sNfL levels were used for our analysis. Subjects missing NfL levels at the timepoint were removed from the analysis for that time point. sNfL at baseline was averaged with sNfL at year one, with sNfL at year two and sNfL at year one, two, and three were averaged. Analyses were performed with these different averaged values.
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