Samples were thawed at room temperature and centrifuged at 10,000 x g for 10 minutes. For cohort 1, samples were measured using a pre‐commercial Neurology 4‐plex E kit (Quanterix) that measures Aβ1‐42, Aβ1‐40, GFAP, and NfL simultaneously. This Neurology 4‐plex E was developed in a collaboration between Amsterdam University Medical Centers, and biotechnology companies ADx NeuroSciences and Quanterix.35 For cohort 2, the commercial Neurology 4‐plex E kit (Quanterix) was used, which gave different absolute values than the pre‐commercial assay. In the next freeze–thaw cycle, p‐tau181 was measured in 149 of the 160 participant samples of cohort 1 and in all samples of cohort 2, using the pTau‐181 V2 Advantage kit (Quanterix; same kit batch for both cohorts). All measurements were performed on the Simoa HDx analyzer, according to manufacturer's instructions.
Neurology 4 plex e kit
Manufactured by Quanterix
The Neurology 4-plex E kit is a multiplex assay designed to measure the levels of four different neurological biomarkers in a single sample. The kit utilizes Quanterix's proprietary Single Molecule Array (Simoa) technology to provide highly sensitive and quantitative results. The core function of this product is to enable the simultaneous detection and quantification of these biomarkers, which may be useful for research applications.
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Lab products found in correlation
7 protocols using neurology 4 plex e kit
1
Multiplex Biomarker Measurement for Neurological Disorders
2
Quantification of Neurological Biomarkers
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Plasma and CSF levels of the analytes of interest were determined by commercial SIMOA (Single Molecule Array) assays on an HD-X platform (Quanterix). Levels of Aβ42, Aβ40, GFAP, and NfL were quantified using the Neurology 4-plex E kit (103670; Quanterix) in CSF (400-fold dilution) and plasma (4-fold dilution). Levels of pTau181 were quantified using the pTau181 Advantage kit version 2.1 (104111; Quanterix) in CSF (10-fold dilution) and plasma (4-fold dilution).
Samples were thawed briefly on wet ice. Immediately after thawing, CSF samples used for Aβ40, Aβ42, NfL and GFAP assays were diluted 400x in N4PE CSF Sample Diluent. All calibrators, controls, and samples were then incubated at room temperature for one hour. Samples and internal controls were centrifuged at 10,000 x g for five minutes. All plasma samples were diluted 4x onboard using the provided sample diluent. CSF samples were diluted at the bench and run neat. A four-parameter logistic curve fit, 1/y2 weighted was used for GFAP, NfL and pTau181 while a 5-paramter logistic curve fit, 1/y2 weighted was used for Aβ40 and Aβ42. Two control samples of known concentration (high-control and low-control) provided in each kit as well as two internal controls of pooled CSF and plasma were included as quality control.
Samples were thawed briefly on wet ice. Immediately after thawing, CSF samples used for Aβ40, Aβ42, NfL and GFAP assays were diluted 400x in N4PE CSF Sample Diluent. All calibrators, controls, and samples were then incubated at room temperature for one hour. Samples and internal controls were centrifuged at 10,000 x g for five minutes. All plasma samples were diluted 4x onboard using the provided sample diluent. CSF samples were diluted at the bench and run neat. A four-parameter logistic curve fit, 1/y2 weighted was used for GFAP, NfL and pTau181 while a 5-paramter logistic curve fit, 1/y2 weighted was used for Aβ40 and Aβ42. Two control samples of known concentration (high-control and low-control) provided in each kit as well as two internal controls of pooled CSF and plasma were included as quality control.
3
Quantification of Plasma Biomarkers
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Plasma samples from TRIAD, WRAP and SPIN were analyzed at the Department of Psychiatry and Neurochemistry, University of Gothenburg. Plasma Aβ42/40, GFAP and NfL were quantified using the commercial Neurology 4-plex E kit (#103670, Quanterix). Plasma pTau231 and pTau181 were analysed using in-house Simoa assays developed at the University of Gothenburg18 (link)
38 (link), except in WRAP where plasma pTau181 was quantified by the commercial Advantage V2.1 kit (#104111, Quanterix) 24 (link).
38 (link), except in WRAP where plasma pTau181 was quantified by the commercial Advantage V2.1 kit (#104111, Quanterix) 24 (link).
4
Comprehensive Metabolic and Neurological Biomarker Profiling
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Approximately 2 wk following Visit 1, subjects returned to the KU Clinical Translational Science Unit (CTSU) for visit 2 following an overnight fast. Blood samples were analyzed using a lipid panel (Quest Diagnostics) to determine cholesterol HDL, LDL, LDL/HDL, and triglyceride values. Blood was collected into serum separator tubes and processed to generate plasma and serum, which was frozen at −80°C until further analysis. Glucose was measured using a colormetric assay (Sigma-Aldrich, St. Louis, MO) and insulin was measured using ELISA (Alpco Diagnostics). Amyloid beta 42 (Aβ42), Amyloid beta 40 (Aβ40), neurofilament light (NFL), and glial fibrillary acidic protein (GFAP) were measured on a Simoa HD-X machine in plasma using the Neurology 4-Plex E kit (Quanterix).
5
Plasma Biomarker Quantification Using Simoa
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EDTA plasma samples were fractionated from overnight fasted blood. Plasma samples were stored at –80°C prior to thawing for measurement of GFAP, Aβ1 - 40, and Aβ1 - 42 concentrations using the single-molecule array (Simoa®) platform using the Neurology 4-Plex E kit (QTX-103670, Quanterix, Billerica, MA) wherein calibrators were run in duplicates and samples were run in singlicates. Quality control (QC) was achieved by assessing the levels of the positive controls included in the Simoa kits. The analytical lowest limit of quantification was 11.6 pg/ml for GFAP, 1.51 pg/ml for Aβ1 - 42 and 4.08 pg/ml for Aβ1 - 40. The average % CV of the two quality controls were 1.68% and 1.46% for GFAP, 1.28% and 1.06% for Aβ1 - 42 and 0.2% and 2.19% for Aβ1 - 40, respectively.
6
Plasma Biomarker Measurement Protocol
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Briefly, participants underwent venipuncture under overnight‐fasting conditions between 2006 and 2009. Plasma was then centrifuged for 10 minutes at 1800g within 2 hours. Then, in polypropylene tubes, plasma was aliquoted in 0.5‐mL aliquots and stored at ‐80 °C until use. Plasma markers were analyzed in 2021. Aβ40, Aβ42, NfL, and GFAP were all assessed using the Neurology 4‐plex E kit (Quanterix).
30 (link) P‐tau181 was assessed using the V2 Advantage kit (Quanterix). Measurements were performed according to manufacturer's instructions, using automated sample dilution on board of the Simoa HD‐X analyzer. The Neurology 4‐plex E kit was run in singlicates, and the p‐tau181 V2 kit was run in duplicates. We calculated Aβ42/40, to adjust for between‐person differences in production rates and to correct for preanalytical sample handling effects.
31 (link) All plasma markers were Z score standardized for analysis.
30 (link) P‐tau181 was assessed using the V2 Advantage kit (Quanterix). Measurements were performed according to manufacturer's instructions, using automated sample dilution on board of the Simoa HD‐X analyzer. The Neurology 4‐plex E kit was run in singlicates, and the p‐tau181 V2 kit was run in duplicates. We calculated Aβ42/40, to adjust for between‐person differences in production rates and to correct for preanalytical sample handling effects.
31 (link) All plasma markers were Z score standardized for analysis.
7
Serum Biomarkers in the AIBL Cohort
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Participants were from the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL) cohort. Participant exclusion criteria are described in detail elsewhere. 21 was 5.58%. Aβ1-40, Aβ1-42, GFAP, and NfL were measured using the Neurology 4-Plex E kit (QTX-103670, Quanterix, Billerica, MA), where calibrators were run in duplicates and samples in singlicates. Average CV% of previous batches run in duplicate in our laboratory for Aβ1-40, Aβ1-42, GFAP, and NfL were 1.56%, 2.91%, 3.26%, and 3.20%, respectively. Quality control (QC) was attained by assessing the levels of the positive controls provided in the Simoa kits. The analytical lowest limit of quantification was 0.338 pg/mL for p-tau181, 4.08 pg/mL for Aβ1-40, 1.51 pg/mL for Aβ1-42, 11.6 pg/mL for GFAP, and 1.6 pg/mL for NfL. The average %CV of the two quality controls was 1.7% and 6.6% for p-tau181, 0.2% and 2.19% for Aβ1-40, 1.28% and 1.06% for Aβ1-42, 1.68% and 1.46% for GFAP, and 0.17% and 1.48% for NfL, respectively.
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