Elisapro kit

Supernatants from WBAs were thawed, diluted, and assayed for IFN-γ production using an ELISA^PRO kit (Mabtech, Sweden). The assay kit has an effective range of 3.16 to 3,160 pg/ml and was performed according to the manufacturer's instructions.
Since higher concentrations of IFN-γ were expected, supernatants were diluted prior to performing the assay. Taking the respective dilution factor into account, concentrations between 31.6 and 10,000 pg/ml and between 158 and 50,000 pg/ml, respectively, could be measured in 12-h and 7-day stimulation assays.

IL-10 and IL-17 levels were determined in serial serum samples, which were collected daily from the time of admission to discharge, using the human IL-10 enzyme linked immunosorbent assay (ELISA) PRO kit (Mabtech, Sweden) and LEGEND MAX™ Human IL-17A ELISA Kit (BioLegend, USA). The ELISAs were performed and results were interpreted according to the manufacturer’s instructions.

Evaluation of IL-2 concentration was assayed in supernatants of patients PBMC transfected O/N with L-siRNA and L-siRNA-Sig10L at the dose of 80 pmols of siRNA, then treated with Dynabeads Human T-activator CD3/CD28 beads (Invitrogen). After the O/N transfection, cells were washed by centrifugation, seeded 2.5 × 10⁵ per well in 96-well flat bottom plates in complete RPMI medium, then activated with anti-CD3/CD28 beads at the bead-to-cell ratio of 1:50 and then incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 5 days (7 (link)). At the end of the incubation period, supernatants and cells were collected and separated by centrifugation at 1,200 rpm for 5 min. Supernatants IL-2 concentration was estimated by means of the human IL-2 ELISA development kit (Elisa^Pro Kit, Mabtech, Nacka Strand, Sweden) according to the instructions of the manufacturer. Plates were read at 405 nm by Bench-mark Plus microplate spectrophotometer (Bio-Rad, CA). The evaluation was performed on at least triplicate biological determinations. Demographic and clinical characteristics of the patients are shown in Table S2.