Neurofilament light chain (NfL) levels in blood sera were quantified using the Simoa NF-light Advantage Kit (103186, Quanterix, USA). Sera were diluted 1:40 (v/v) in sample dilution buffer and measured on a Simoa HD-1 Analyzer (Quanterix) following manufacturer’s instructions. NfL serum concentration was interpolated from standard curves and mean values from duplicate measurements were plotted.
Simoa nf light advantage kit
Manufactured by Quanterix
Sourced in United States
The Simoa NF-Light Advantage Kit is a lab equipment product manufactured by Quanterix. It is designed to detect and quantify Neurofilament Light (NF-Light) protein levels in biological samples using the Simoa technology.
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44 protocols using simoa nf light advantage kit
1
Quantification of Neurofilament Light Chain
2
Quantification of Neurofilament Light Protein
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Serum samples were collected monthly and directly stored at −20°C until after preparation because NfL protein is stable during freezing process.24 (link),25 (link) sNfL measurement was performed using the Advantage NF-Light Singleplex Kit and prepared as defined in the manufacturer's instructions (Quartered, Lexington, MA, Datasheet Quanterix: SIMOA NF-Light Advantage Kit). The SIMOA Human Advantage NfL assay is a digital immunoassay for the quantitative determination of NfL and was ran on a Simoa HD-1 instrument (Quanterix).26 (link) The antibodies and calibrators used in the assays have been developed by Uman Diagnostics (Sweden) and described previously in their plate-based enzyme-linked immunosorbent assay.27 (link) Previous reports proved that the newly developed SIMOA technique is the most sensitive to detect NfL even in lowest concentrations.28 (link) Calibrators (8 calibrator points) and diluted serum samples (dilution 1:4) were measured in duplicates. Sample dilution was calculated using the instrument. The lower threshold of quantification was 0.775 pg/mL. Both the mean intraassay coefficient of variation of duplicates and the mean interassay coefficient of variation were <10%.
3
Cerebellar Metabolites and Serum Neurofilament Light Quantification
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All participants underwent identical MR procedures using a Siemens 1.5T magnetic resonance imaging (MRI) scanner. 1 H-MRS measurements were then performed to determine the value of N-acetyl aspartate (NAA)/creatine (Cr), and choline complex (Cho)/Cr in local cerebellar metabolites [16] . The multiple-voxel 1 H-MRS sequence was acquired with the following parameters: repetition time (TR) = 4,280 ms, echo time (TE) = 135 ms, and voxel size = 6.3 mm × 6.3 mm × 15 mm. We selected the target as the cerebellum, which includes the bilateral cerebellar hemispheres, the dentate nucleus, and the cerebellar vermis, in sequence by voxel. The local cerebellar metabolites consisted of the values of NAA/Cr and Cho/Cr, as detected via a proton MR wave.
Sn Levels (Dup: Abstract ?) Serum samples were taken from all participants and collected according to standardized local procedures. Then, the serum samples were aliquoted and immediately stored at -80°C. sNfL concentrations were detected using a single-molecule (Simoa) array method and then quanti ed on the Simoa HD-1 Analyzer platform (GBIO, Hangzhou, China), using a Simoa NF-light Advantage Kit (Quanterix, Massachusetts, USA) [17] (link). All sNfL concentrations were within the linear ranges of this assay.
Sn Levels (Dup: Abstract ?) Serum samples were taken from all participants and collected according to standardized local procedures. Then, the serum samples were aliquoted and immediately stored at -80°C. sNfL concentrations were detected using a single-molecule (Simoa) array method and then quanti ed on the Simoa HD-1 Analyzer platform (GBIO, Hangzhou, China), using a Simoa NF-light Advantage Kit (Quanterix, Massachusetts, USA) [17] (link). All sNfL concentrations were within the linear ranges of this assay.
4
CSF NfL Quantification by Simoa Assay
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NfL levels were quantitatively determined in CSF samples using the Simoa NF‐light Advantage kit (Quanterix, #103186) following the manufacturer’s instructions. CSF samples were diluted 1:10 in sample dilution buffer and mixed with Simoa detector reagent and bead reagent, following an incubation at 30°C for 30 min, shaking at 800 rpm. Plates were washed with Simoa washing buffer A and SBG reagent from the kit was added. Following a 10 min incubation at 30°C and shaking at 800 rpm, plates were washed twice and sample beads were resuspended in Simoa wash buffer B. NfL concentrations were measured after a 10 min drying at RT using the Simoa DH‐1 analyzer (Quanterix).
5
Standardized Serum NfL Measurement Protocol
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Blood was collected and processed, and serum was stored according to a standardized protocol as previously described.11 (link) NfL measurement in serum samples was performed in one laboratory by experienced laboratory analysts blinded to clinical and genetic information. Serum NfL was measured in 2 batches using the Simoa NF-Light Advantage Kit (Quanterix; Billerica, MA) on a Simoa HD-1 Analyzer instrument for the first batch (January 2018) or a Simoa HD-X for the second batch (March 2022), according to the manufacturer's instructions and using identical protocols. In each batch, longitudinal samples from the same participant were included in the same run. To account for batch effects in NfL measurements, a subset of the samples (n = 109 from 54 participants) was measured in duplicate in both batches (eFigure 1, links.lww.com/WNL/C981 ), and linear regression transformation factors were derived and applied to batch 1 data (eFigure 2), obtaining optimal transformation outcomes (eTable 1). See eMethods for details on the transformation. All presented data are transformed data. The median number of NfL measurements per participant was 4 (interquartile range [IQR] 3–6), with a median time interval between samples of 1.2 years (IQR 1.0–2.0).
6
Serum NfL Quantification from Murine Blood
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Peripheral blood was collected from the tail vein by making a small incision in the lateral tail vein after swabbing with a disinfectant wipe. Two to three drops of blood (approx. 30 µL) were collected in a sterile Eppendorf tube. The tubes were maintained at room temperature for 30 to 60 minutes to allow coagulation and then centrifuged at 2000g for 25 minutes. The upper serum layer was collected and kept at −80°C until analysis. Before analysis, samples were thawed at room temperature and centrifuged at 10,000g. Subsequently, serum NfL concentrations were determined in accordance with manufacturer guidelines using Simoa NF-light advantage kit (Quanterix, MA).
7
Quantifying Biomarkers in Dog Serum
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Concentrations of neurofilament light chain (NFL), TAU proteins, and Aβ42 peptide in blinded dog serum samples were measured using Simoa digital ELISA, using an HD-1 Analyzer (Quanterix, Boston, USA). For the analysis, Simoa™ NF-Light Advantage Kit, Simoa™ Mouse TAU Discovery Kit, and Simoa™ Aβ-42 Advantage Kit were used (Quanterix, Boston, USA). Briefly, frozen dog serum samples were melted, centrifuged for 10 min at 25°C, and 20,000 × g, and supernatants were transferred onto Simoa sample plates together with calibrators for NFL, mouse TAU, and Aβ42 kits (parts of kits). Each measurement was done in duplicate according to the manufacturer's recommendations. Concentrations were calculated using Simoa™ HD-1 instrument software.
8
Quantifying Neurofilament Light in Plasma
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Plasma samples were collected from each patient and frozen at −20°C. NfL concentrations were measured with Simoa NF-Light Advantage Kit (Quanterix, Lexington, MA) according to the manufacturer’s instructions. The assay was carried out on Simoa HD-X Analyser (Quanterix Corporation, Lexington, MA, USA). We analysed all samples in duplicate within one assay. The mean intra-assay coefficient of variation of duplicate determinations for concentration ranged from 3% to 6%.
9
Serum Neurofilament Light Protein Quantification
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Serum NfL was measured using a commercial Simoa® NF-light™ Advantage Kit on a HDX analyzer (Quanterix, Billerica, MA, USA) based on single-molecule arrays and the simultaneous counting of single captured microscopic beads carrying antibody complexes. The analytical sensitivity of this technique enables a reliable measurement of low NfL concentrations in blood samples [17 (link)]. Advanced SIMOA NfL-kits (Quanterix, MA, USA) developed for human research use were utilized according to the manufacturer’s instructions, since there is a high cross-reactivity for rats.
10
Quantification of Phosphorylated Neurofilament Light
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Samples from the stored aliquots were shipped to prismCDX Co., Ltd. (Hwaseong, Korea) for the analysis of phosphorylated NfL (pNfL) levels using a Simoa NF-light Advantage Kit (Quanterix, Billerica, MA, USA) with a Simoa HD-1 Analyzer (Quanterix), according to the manufacturer’s instructions (https://www.quanterix.com/resources/publications-posters ). For calibration, a combination of monoclonal antibodies and purified bovine NfL was used. Concentrations of pNfL were measured, in units of pg/mL, while demographic and clinical data were blinded. Analytical sensitivity was <1.0 pg/mL. All samples tested were within the assay’s dynamic range, with the coefficient of variation ranging from 4.2% to 7.4% for measurement. The lower limit of quantification was 10.0 pg/mL, and the upper limit of quantification was 498.0 pg/mL. All samples were measured within the range spanned by the limits of quantification. The reliability of quantification was high (R2=0.999 of calibration curve).
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