Multiplex elisa assay

Cell-free cell culture supernatant samples were stored at −20°C until analyzed. The system used was a multiplex ELISA assay manufactured by Meso Scale Discovery (MSD, Gaithersburg, MD, USA) containing 10 individual ELISAs per well. Supernatants were tested with a U-PLEX Proinflammatory Panel1 Human Kit [a multiplex 96-well ELISA plate based assay that contained primary antibodies to IL-12, IL-10, TNFα, IL-6, IL22, interleukin 1 receptor antagonist (IL-1Ra), interferon alpha 2a (IFNα2a), interferon beta (IFNβ), interferon gamma (IFNγ), and IL-1β] per manufacturer’s recommendations. Briefly, the MSD plex assays were run as follows. Calibration curves were prepared in the supplied assay diluent for human serum, with a range of 40,000–1.2 pg/ml, depending on the cytokine. Arrays were preincubated with 25 µl per well of assay diluent for 30 min. After the pre-incubation, 25 μl sample or calibrator was added in duplicate to the appropriate wells. The array was then incubated at room temperature for 2 h with primary antibody. The array was washed with PBS plus 0.05% Tween 20, and 25 µl detection antibody reagent was added. After 2 h of incubation at room temperature with secondary antibody, the array was washed, and the detection buffer was added. Results were read with a QuickPlex SQ 120. Sample cytokine concentrations were determined using MSD Discovery Workbench 4.0 software.