Verikine human interferon alpha elisa kit

5x10⁵ PBMCs per well were seeded in U-bottom 96-well tissue culture plates (Greiner Bio-One, Austria) and stimulated with 10 ng/ml lipopolysaccharides (LPS) derived from Escherichia coli O55:B5 (Sigma-Aldrich, MO, USA), 10⁶/ml heat-killed Staphylococcus aureus, 10⁶/ml heat-killed Candida albicans, R848 (InvivoGen), or heat-inactivated(30 minutes, 60°C) SARS-CoV-2 Wuhan-Hu-1 strain (NRW-42 isolate, GISAID accession number: EPI_ISL_425126) (35 (link)) with 1 µg/ml soluble purified mouse anti-human CD28 (BD Biosciences, New Jersey, USA), for 24 hours and 7 days in the presence of 10% pooled human serum. A control condition without any stimulant was included. Cells were incubated at 37°C with 5% CO2. Stimulations with R848 and SARS-CoV-2 were performed later with cryopreserved cells, while the rest of the stimulations were done with freshly isolated PBMCs. Concentrations of TNF-α, IL-6, IL-1β, IL-1Ra, and IFN-γ in culture supernatants were determined using DuoSet ELISA kits (R&D Systems, MN, USA). IFNα concentrations were determined using VeriKine Human Interferon Alpha ELISA Kit (PBL Assay Science, NJ, USA) according to the manufacturer’s instructions. Supernatants from 7 days stimulations were used for IFN-γ, and the remaining cytokines were measured in 24-hour supernatants.

Production of secreted cytokines in PBMC culture supernatants was measured using ELISA. IL-1β, IL-6, TNF, CXCL10, interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1Ra), and interferon-gamma (IFN-γ) concentrations were determined with duoSet ELISA kits (R&D Systems). The VeriKine Human Interferon Alpha ELISA Kit (PBL Assay Science) was used to measure interferon-alfa (IFN-α). Supernatants from 7-day cultures were used for IFN-γ measurements, whereas the remaining cytokines were measured in 24-hour culture supernatants. Plasma concentrations of Interferon Stimulated Gene 15 (ISG15) were measured using the Human ISG15 ELISA kit from Elabscience (Elabscience). All tests were performed according to the instructions of the manufacturer.