Human quantikine immunoassays

Blood concentrations of IL-1β, IL-18, and tumor necrosis factor-α (TNF-α) were measured by the Human Quantikine^® Immunoassays (R&D Systems, Minneapolis, MN, USA). IL-6 was measured on the Siemens 150.
Immulite 2000 XPI system using the Siemens IL-6 reagent (Siemens, Erlangen, Germany). HMGB1 was measured by an ELISA obtained from IBL International (Hamburg, Germany). To detect low cytokine levels in cell culture supernatants, for IL-1β the Human IL-1 beta/IL-1F2 DuoSet ELISA (R&D Systems) was used, whereas IL6 and TNF-α were measured by the Human Quantikine^® Immunoassays (R&D Systems, Minneapolis, MN, USA).

Markers of microbial and fungal translocation (soluble CD14, sCD14, 1,3-β-D-Glucan), epithelial barrier integrity (intestinal fatty binding acid protein, I-FABP), inflammation (C reactive protein, CRP), and adipose tissue inflammation (sCD163, monocyte chemoattractant protein-1, MCP-1 and adiponectin) were measured in plasma using commercial enzyme-linked immunosorbent assays (ELISA) according to their respective package inserts. Human Quantikine Immunoassays were used for sCD14, sCD163, CRP, MCP-1, adiponectin (R&D systems, Minneapolis, Minnesota, USA), Hycult Biotech (Uden, The Netherlands) for I-FABP and MyBioSource (San Diego, CA, USA) for 1,3-β-D-Glucan. Plasma samples were diluted 250-fold for sCD14, 25-fold for sCD163, 3-fold for I-FABP and MCP-1, and 150-fold for CRP and adiponectin. For 1,3-β-D-Glucan, plasma was used neat. Assays were read at 450nm (Epoch, BioTek, Winooski, Vermont, USA) and analyzed using the Gen5 software, version 2.07. Wavelength correction was applied by resting the optical density at 570nm. Samples were run in duplicate for all assays.