Human il 6 quantikine hs elisa kit

As previously described [18 (link),25 (link)], single morning blood samples were collected between 10:00 a.m. and 12:00 p.m., transferred to EDTA-containing tubes (3 per patient), and refrigerated until centrifugation (within 3 h) for plasma isolation, which was kept in deep freeze (−80 °C). Plasma cortisol was measured using the ELISA technique (Cusabio Human cortisol ELISA kit, cfb-e05111h, Cusabio Technology LLD, Houston, Texas, USA). The inter- and intra-assay coefficients of variation were 23.1% and 16.1%, respectively. The lower detection limits for cortisol were 0.02 ngr/mL.
Plasma IL-6 was measured using the ELISA technique (Human IL-6 Quantikine HS ELISA kits, R&D Systems Europe, Abington, UK). For the IL-6 determination, the inter-assay coefficients of variation were 13.9%, the intra-assay coefficients of variation were 11.04 and the lower detection limits was 0.133 pg/mL.

The measurement of serum cytokines concentrations was determined with the usage of a quantitative commercial enzyme-linked immunosorbent assay (ELISA) technique, following the manufacturers’ recommendations. In brief, the serum concentration of IL-2 was measured using a Human IL-2 Quantikine ELISA Kit (sensitivity, 7 pg/mL; R&D Systems, Minneapolis, MN, USA); the serum concentration of IL-4 was determined using the Human IL-4 Quantikine HS ELISA Kit (sensitivity, 0.22 pg/mL; R&D Systems, USA); the concentration of serum IL-6 was measured using the Human IL-6 Quantikine HS ELISA Kit (sensitivity, 0.11 pg/mL; R&D Systems, USA); the serum concentration of IL-10 was determined using the Human IL-10 Quantikine HS ELISA Kit (sensitivity, 0.17 pg/mL; R&D Systems, USA), and the concentration of serum IFN-gamma was determined using the Human IFN-γ Quantikine ELISA Kit (sensitivity, 8 pg/mL; R&D Systems, USA). The results were measured with an automatic reader VICTOR3 (Perkin Elmer, Waltham, MA, USA); that action is based on the measurements of the light absorbance of the tested material and its comparison with control samples of known concentration. The WorkOut computer program, working with the reader on the basis of known concentrations, plotted linear curves on the basis of which the concentration of cytokines in the tested samples was calculated.

Blood samples were collected after a 12 h overnight fast and 48 h alcohol abstinence, from an antecubital vein into vacutainer tubes containing EDTA. Plasma total cholesterol and plasma TG concentrations were measured using enzymatic assays [36 (link),37 (link)]. Infranatant (d > 1.006 g/mL) with heparin-manganese chloride was used to precipitate very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), and then to determine high-density lipoprotein (HDL)-cholesterol (HDL-C) concentrations [38 (link)]. Plasma C-reactive protein (CRP) was measured by nephelometry (Prospec equipment, Behring Diagnostic, Westwood, MA, USA) using a sensitive assay, as described previously [39 (link)]. Plasma concentrations of interleukin-6 (IL6) and tumor necrosis factor-α (TNF-α) were measured with high-sensitivity ELISA kits including: Human IL6 Quantikine HS ELISA Kit Minneapolis, MN, USA (R&D Systems, Minneapolis, MN, USA (HS600B)) and Human TNF-alpha Quantikine HS ELISA Kit (R&D Systems (HSTA00D)) [39 (link)]. Fasting insulin levels were measured using radioimmunoassay with polyethylene glycol separation [40 (link)]. Fasting glucose levels were enzymatically measured [41 (link)].

The blood samples were centrifuged for 10 min at 1500× g to obtain plasma samples which were kept at −80 °C for subsequent cytokine analysis. The levels of IL-8, IL-18, IL-23, IL-4, TGF-β, IL-22, IL-1β, IL-10, IL-15, CCL20, CCL2, CCL5, CX3CL1, and BDNF were measured by DuoSet^® ELISA Kits (R&D Systems, Minneapolis, MN, USA). The IL-13 levels were measured by the Human IL-13 ELISA kit (Invitrogen, Thermo Fisher, Waltham, MA, USA). High-sensitivity kits were required to evaluate IL-6 (Human IL-6 Quantikine HS ELISA Kit, R&D Systems, Minneapolis, MN, USA). The concentrations of TNF-α, IL-12p70, IL-17A, and IFN-γ were measured with cytokine 6-plex Panel 1 (TNF-α, IL-12p70, IL-17A, IL-10, IL-6, and IFN-γ) (IFN-γ, IL-6, IL-10, IL-12p70, IL-17A, TNF-a) (Quanterix Corp., Billerica, MA, USA) using SIMOA SR-X equipment (Quanterix Corp., Billerica, MA, USA). Additional measurements for IL-17A were performed with the IL-17A 2.0 Advantage Assay using SIMOA™ HD-X equipment (Quanterix Corp., Billerica, MA, USA).

Plasma was obtained from whole blood, drawn in EDTA tube, by centrifuging at 400xg, for 10 min at room temperature. Plasma samples from IgAN patients and HC were then analyzed with enzyme-linked immunosorbent assay (ELISA) to determine the concentration of the following factors; monocyte chemoattractant protein (MCP-1) (Human CCL2/MCP-1 Quantikine ELISA Kit, R&D Systems, USA), soluble CD14 (Human CD14 Quantikine ELISA Kit, R&D Systems), soluble CD40L (Human CD40 Ligand/TNFSF5 Quantikine ELISA Kit, R&D Systems), B cell activating factor (BAFF) (Human BAFF Quantikine ELISA Kit, R&D Systems), IL-6 (Human IL-6 Quantikine HS ELISA Kit, R&D Systems), Fractalkine (Human CX3CL1/Fractalkine Quantikine ELISA Kit, R&D Systems) and MIP-1 (Human CCL3/MIP-1 alpha Quantikine ELISA Kit, R&D Systems). Catalog number for ELISA kits are shown in S2 Table. The samples were assayed in duplicates and the average values were considered for further analysis. The optical density (OD) in blank well was subtracted from the OD values in samples before calculation of concentrations.