Meso Scale Discovery multiplex assay

V-PLEX Custom Human Cytokine10-plex kits for Proinflammatory Panel1 and Chemokine Panel 1 (K151A0H-2, for IL-1β, IL-6, IL-8, IL-10, TNF, CCL2, CXCL10) and U-PLEX human Interferon Combo SECTOR (K15094K-2, for IFN-α2a, IFN-β, IFN-γ, IFN-λ1) were purchased from Mesoscale Discovery (MSD). The lyophilized cocktail mix calibrators for Proinflammatory Panel 1, Chemokine Panel 1, and 4 calibrators for U-PLEX Biomarker Group 1 (Calibrator 1, 3, 6, 9) were reconstituted in provided assay diluents respectively. U-PLEX plates were coating with supplied linkers and biotinylated capture antibodies according to manufacturer’s instructions. Proinflammatory cytokines and chemokines in supernatant collected at 24 hours after stimulation were detected with pre-coated V-PLEX and interferons in 6 hour supernatant were measured by coated U-PLEX plates. The assays were performed according to manufacturer’s protocol with overnight incubation of the diluted samples and standards at 4°C. The electrochemiluminescence signal (ECL) were detected by MESO QuickPlex SQ 120 plate reader (MSD) and analyzed with Discovery Workbench Software (v4.0, MSD). The concentration of each sample was calculated based on the four-parameter logistic fitting model generated with the standards (concentration was determined according to the certificate of analysis provided by MSD). log₁₀ values of measured concentrations of IL-1β, IL-6, IL-8, IL-10, TNF, IFN-β, IFN-γ, CXCL10 were used for the principle component analysis. log₁₀ IgG titers (half maximal effective concentration, EC₅₀) were used for the color overlay.