Plasma (EDTA) levels of HGF, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor-2, and caspase-8 were analyzed by the proximity extension assay technique using the Proseek Multiplex CVD96✕96 reagents kit (Olink Bioscience) at the Clinical Biomarkers Facility, Science for Life Laboratory, Uppsala. The coefficients of variation for intra-assay variation (within-run) and interassay variation (between-run) for HGF, TRAIL receptor-2, and caspase-8 are 8% and 13%, 7% and 10%, and 5% and 10%, respectively, and the analytical ranges were 15.3-31,250 pg/mL, 0.2-7,812 ng/mL, and 3.8-62,500 pg/mL, respectively. Data analysis was performed by a preprocessing normalization procedure using Olink Wizard for GenEx (Multid Analyses). All data are presented as arbitrary units (AUs). General calibrator curves to calculate the approximate concentrations as well as technical information about the assays are available on the Olink homepage.
Proseek multiplex cvd96 96 reagents kit
Manufactured by Olink
Sourced in Sweden
The Proseek Multiplex CVD96×96 reagents kit is a laboratory equipment product designed to facilitate the simultaneous measurement of up to 96 protein biomarkers from 96 samples. The kit provides the necessary reagents and materials for the analysis of cardiovascular disease-related proteins using the Proseek Multiplex technology.
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6 protocols using proseek multiplex cvd96 96 reagents kit
1
Plasma Biomarkers of Apoptosis and Angiogenesis
2
Plasma Biomarkers Profiling by PEA
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Plasma levels of renin and IL-6 were analyzed by the Proximity Extension Assay (PEA) technique using the Proseek Multiplex CVD96×96 reagents kit (Olink Bioscience, Uppsala, Sweden) at the Clinical Biomarkers Facility, Science for Life Laboratory, Uppsala. Oligonucleotide-labeled antibody probe pairs were allowed to bind to their respective targets present in the plasma sample and addition of a DNA polymerase led to an extension and joining of the two oligonucleotides and formation of a PCR template. Universal primers were used to pre-amplify the DNA templates in parallel. Finally, the individual DNA sequences were detected and quantified using specific primers by microfluidic real-time quantitative PCR chip (96.96, Dynamic Array IFC, Fluidigm Biomark). The chip was run with a Biomark HD instrument. The mean coefficients of variance for intra-assay variation and inter-assay variation are 7 and 13 % for renin, and 8 and 10 % for IL-6, respectively. All samples were analyzed in the same run. Data analysis was performed by a preprocessing normalization procedure using Olink Wizard for GenEx (Multid Analyses, Sweden). All data are presented as arbitrary units (AU). General calibrator curves to calculate the approximate concentrations as well as technical information about the assays are available on the Olink homepage (http://www.olink.com ).
3
Plasma MMP Biomarkers Profiling
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Circulating levels of plasma levels of MMP‐1, ‐3, ‐7, ‐10 and ‐12 were assessed in 551 patients using the proximity extension assay technique using the Proseek Multiplex CVD96×96 reagents kit (Olink Bioscience, Uppsala, Sweden) at the Clinical Biomarkers Facility, Science for Life Laboratory, Uppsala, as previously described.17
4
Galectin-3 Levels in Children
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Non-fasting venous serum samples were drawn from 172 children and stored at − 70 °C until analysis. The samples were collected in conjunction with the DXA measurement. Galectin-3 was analyzed by the Proximity Extension Assay technique using the Proseek Multiplex CVD 96 × 96 reagents kit (Olink Bioscience, Uppsala, Sweden) at the Clinical Biomarkers Facility, Science for Life Laboratory, Uppsala, as previously described [25 (link), 26 (link)]. The intra- and inter-assay variations were 9 and 12%, respectively. Data are presented as arbitrary units (AU). Values can be transformed to actual concentrations on the Olink Bioscience website. Two samples were excluded due to technical problems at the analysis.
5
Quantifying Inflammatory Markers in Plaque Homogenates
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Aliquots of 50 μL of plaque homogenate were centrifuged at 13,000 g for 10 min. Twenty-five microliters of the supernatant were removed and used for measuring interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), tumor necrosis factor-α, and interferon-γ. The procedure was performed according to the manufacturer's instructions (Human Cytokine/Chemokine Immunoassay, Millipore Corp., Danvers, MA, USA) and analyzed with Luminex 100 IS 2.3 (Luminex Corp., Austin, TX, USA). Matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-9, and MMP-10 were analyzed in 25 μL of the supernatant using the Mesoscale human MMP ultrasensitive kit (Mesoscale, Gaithersburg, MD, USA), according to the manufacturer's instructions. Vascular endothelial growth factor A (VEGF-A), osteoprotegerin (OPG), and C-C motif chemokine 3 (CCL3) and CCL20 levels were analyzed by the Proximity Extension Assay technique using the Proseek Multiplex CVD96×96 reagents kit (Olink Bioscience, Uppsala, Sweden) as previously described [14 (link)]. Data analysis was performed by a preprocessing normalization procedure using Olink Wizard for GenEx (Multid Analyses, Gothenburg, Sweden). VEGF-A, CCL3, CCL4, and CCL20 levels are presented as arbitrary units. Results were normalized to the wet weight of the plaque (Table 1 ).
6
Cardiovascular Biomarkers Analysis Protocol
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Plasma levels of low density lipoprotein cholesterol (LDL), HDL, TG and glucose were determined within 1 week of CV examination by standard methods on a Roche-Modular Autoanalyzer (Milan, Italy). Glycosylated hemoglobin (HbA1c) was measured by high-performance liquid chromatography and standardized against Diabetes Control and Complications Trial (DCCT ) standard. Plasma levels of biomarkers reflecting inflammation (IL-6 and IL-18) and extracellular matrix proteolysis (MMP-12) were analyzed by the Proximity Extension Assay (PEA) technique using the Proseek Multiplex CVD96×96 reagents kit (Olink Bioscience, Uppsala, Sweden) at the Clinical Biomarkers Facility, Science for Life Laboratory, Uppsala as previously described [9 (link), 26 (link)]. All samples were analyzed in the same run. Data analysis was performed by a preprocessing normalization procedure using Olink Wizard for GenEx (Multid Analyses, Sweden). Values are presented as arbitrary units (AU). Data regarding intra- and inter-assays variations as well as general calibrator curves to calculate the approximate concentrations are available on the OLINK homepage (http://www.olink.com ).
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