Blood was drawn from participants concurrent with CMR, and samples were processed for serum and plasma on the same day using standard protocols and stored at − 80 °C until assays were performed. Biomarkers of inflammation (interleukin-6 [IL-6], soluble cluster of differentiation 14 [sCD14], galectin-3), ventricular and cellular stress (N-terminal prohormone of brain natriuretic peptide [NT-proBNP], growth differentiation factor-15 [GDF-15]), tissue remodeling (tissue inhibitor of metalloproteinase-2 [TIMP-2], matrix metalloproteinase-2 [MMP-2]) and tissue injury (high-sensitivity troponin I [hsTnI]) were measured in serum and plasma at central laboratories. Serum levels of IL-6 were measured by electrochemiluminescence (Meso Scale Discovery V-PLEX), and the following markers were measured by enzyme-linked immunosorbent assay: sCD14 (R&D Systems Quantikine), GDF-15 (R&D Systems Quantikine), TIMP-2 (R&D Systems Quantikine), MMP-2 (R&D Systems Quantikine), and NT-proBNP (Abbott Architect i2000sR) in serum, and galectin-3 (Abbott Architect i2000sR) and hsTnI (Abbott Architect i2000sR) in plasma.
Quantikine
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany, France
The Quantikine is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kit developed by R&D Systems. It is designed to measure the concentration of specific proteins in a variety of sample types, including cell culture supernatants, serum, and plasma.
Automatically generated - may contain errors
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Quantikine hs, by R&D Systems (14 mentions)
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Synergy ht, by Agilent Technologies (4 mentions)
Duoset elisa kit, by R&D Systems (4 mentions)
Bn 2, by Siemens (4 mentions)
Elisa, by R&D Systems (4 mentions)
Biomek fxp, by Beckman Coulter (3 mentions)
Immulite, by Siemens (3 mentions)
Bnii nephelometer, by Siemens (3 mentions)
Cd3 cd28 beads, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Celltracetm, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Collagenase a, by Roche (2 mentions)
Emax precision microplate reader, by Molecular Devices (2 mentions)
Spectramax m2, by Molecular Devices (2 mentions)
Immulite 2000, by Siemens (2 mentions)
490 protocols using quantikine
1
Biomarker Assessment in Cardiovascular Health
2
Quantifying Inflammatory Biomarkers in Samples
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The serum level of tumor necrosis factor-alpha (TNF-alpha) was measured in duplicate by using a commercially available enzyme immunoassay (Quantikine, R&D Systems Inc., Minneapolis, USA). The sensitivity of this assay was 2.6 pg/ml, the intra-assay coefficient of variation was ±8.8%, and the interassay coefficient of variation was ±16.7%. In addition, the serum level of soluble TNF-alpha receptor I (sTNFr-I) was quantified by ELISA (Quantikine, R&D Systems Inc.), and the serum level of soluble Fas (sFas) was measured by a sandwich enzyme immunoassay (Quantikine, R&D Systems Inc.). Moreover, sFas ligand (sFas L) was quantified with an immunoassay kit (MBL, Tokyo, Japan). In this immunoassay, concomitant measurement of seven sFas L standards with known concentrations (0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 ng/ml) was performed together with patient samples and the detection limit for sFas L was <50 pg/ml. Each assay was performed according to the manufacturer's recommendations [18 (link)].
3
Biomarkers of Cardiorenal Dysfunction
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Fasting venous blood was collected at each visit. Serum creatinine, lipid profile, glycated hemoglobin A1c (HbA1C), and urine albumin to creatinine ratio (ACR) were measured in the local clinical laboratory. Estimated glomerular filtration rate (eGFR) was calculated using the 2009 Chronic Kidney Disease Epidemiology Collaboration creatinine equation (15 (link)). Enzyme-linked immunosorbent assays were performed to measure serum tissue plasminogen activator (Asserachrom, Stago, Reading, United Kingdom), plasminogen activator inhibitor-1 (Asserachrom), von Willebrand factor (vWF) (Asserachrom), and intracellular adhesion molecule (ICAM)-1 (Quantikine, R&D Systems, Abingdon, United Kingdom). Cystatin-C was measured using a particle enhanced turbidimetric immunoassay (Tina-quant, Roche, Germany). Serum high-sensitivity C-reactive protein and lipoprotein(a) were measured (Roche c311 analyzer) and high-sensitivity troponin-I was measured (Abbot, Architect i1000SR). Urine was collected for assessment of interleukin (IL)-18 (Quantikine, R&D Systems).
4
Quantifying Soluble TNF-alpha and Receptor I
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We used the commercially available enzyme immunoassay (Quantikine; R&D Systems, Inc., Minneapolis, USA) to measure soluble TNF-alpha (sTNF-alpha) in duplicate. This enzyme immunoassay has a sensitivity of 2.6 pg/mL, an inter-assay coefficient of variation of +16.7%, and an intra-assay coefficient of variation of +8.8%.
Serum sTNF-alpha receptor I (sTNFr-I) levels were determined using a commercially available enzyme-linked immunosorbent assay system (Quantikine; R&D Systems, Inc.). Blood samples were analyzed within 1 week of collection, and serum was stored at −80°C until use.
Serum sTNF-alpha receptor I (sTNFr-I) levels were determined using a commercially available enzyme-linked immunosorbent assay system (Quantikine; R&D Systems, Inc.). Blood samples were analyzed within 1 week of collection, and serum was stored at −80°C until use.
5
Measurement of Cardiac Biomarkers in Blood Samples
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NT-proBNP was measured with an electrochemiluminescence sandwich immunoassay (Elecsys system 2010®, Roche Diagnostics, Mannheim, Germany). TnT assay was performed with a fourth generation assay (Elecsys 2010®, Roche Diagnostics, Mannheim, Germany).
Blood samples were frozen immediately and stored at −70 °C before measurement of sST2, GDF-15, and OPN. Plasma OPN levels were measured with a sandwich immunoassay by use of a commercially available kit (Quantikine®; R&D Systems, Inc., Minneapolis, MN, USA). Plasma levels of soluble suppression of tumorigenicity 2 (sST2) were determined using a novel high-sensitivity sandwich immunoassay (Presage ST2®; Critical Diagnostics, San Diego, CA, USA). Plasma levels of growth differentiation factor 15 (GDF15) were determined by a sandwich enzyme immunoassay technique (Quantikine®; R&D Systems, Inc., Minneapolis, MN, USA).
Blood samples were frozen immediately and stored at −70 °C before measurement of sST2, GDF-15, and OPN. Plasma OPN levels were measured with a sandwich immunoassay by use of a commercially available kit (Quantikine®; R&D Systems, Inc., Minneapolis, MN, USA). Plasma levels of soluble suppression of tumorigenicity 2 (sST2) were determined using a novel high-sensitivity sandwich immunoassay (Presage ST2®; Critical Diagnostics, San Diego, CA, USA). Plasma levels of growth differentiation factor 15 (GDF15) were determined by a sandwich enzyme immunoassay technique (Quantikine®; R&D Systems, Inc., Minneapolis, MN, USA).
6
Biomarker Measurements in Fasted Serum
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Serum samples were collected after a 12 h overnight fast and immediately stored at −70°C. The samples had not been thawed before the present measurements. Fasting glucose, insulin, and blood lipids were determined by standard methods in the certified clinical chemistry laboratory of the University Hospital. Insulin resistance was estimated by homeostasis model assessment index. The N‐terminal pro‐brain natriuretic peptide levels were determined by a standard chemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany). Activin A levels were determined by enzyme‐linked immunosorbent assay (Quantikine, R&D Systems Europe, Wiesbaden‐Nordenstadt, Germany) and were performed at the Medical Clinic III, Dresden University of Technology, Germany, by investigators that were not aware of patients' characteristics and outcomes. Matrix metalloproteinase 9 (MMP‐9) levels were determined by enzyme‐linked immunosorbent assay (Quantikine, R&D Systems Europe, Abingdon, UK); likewise, adiponectin levels were determined by enzyme‐linked immunoassay (BioVendor GmbH, Heidelberg, Germany).
7
Respiratory Syncytial Virus Infection Cytokine Profile
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BALF was prepared from RSV-infected and uninfected mice on day 1 and day 5 post-infection. Briefly, BALF was obtained from the mice under anesthesia by instilling 0.8 mL of cold PBS into the lungs and aspirating it from the trachea using a tracheal cannula [15 (link)]. The obtained BALF was centrifuged at 160 × g at 4 °C for 10 min. The supernatant was stored at −80 °C until the use for enzyme-linked immunosorbent assay (ELISA). The levels of IFN-α, IFN-β, IFN-γ, and RANTES (CCL5) in BALF were measured using ELISA kits (VeriKine, PBL Assay Science., Piscataway, NJ, USA; Quantikine, R&D Systems, Inc., Minneapolis, MN, USA; Ready-set-go, eBioscience Inc., San Diego, CA, USA; and Quantikine, R&D Systems, Inc., Minneapolis, MN, USA; respectively) according to the manufacturer’s instructions. The lower limits of detection of the kits are 12.5 pg/mL for IFN-α, 1.89 pg/mL for IFN-β, 15 pg/mL for IFN-γ, and 2 pg/mL for RANTES. The intra- and inter-assay coefficients of variation for the ELISA results were less than 10%.
8
Biomarker Assays and Doxycycline Measurements
9
Quantifying Serum Biomarkers by ELISA
10
Quantifying VEGF and sVEGFR-2 in Plasma and Tears
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Before analysis, the plasma and tears were thawed at room temperature. The total protein concentrations of VEGF and sVEGFR-2 were determined by commercially available ELISA methods (Human VEGF, Quantikine; R&D Systems, Inc., Minneapolis, MN, USA; Human sVEGFR-2/KDR/Flk-1, Quantikine; R&D Systems, Inc.) according to the manufacturer's instructions.
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