Duo set elisa kit

Plasma levels of various cytokines (IFN-γ, IL-12p70, IL-4, IL-10, IL-6 and TNF) were measured using sandwich enzyme-linked immunosorbent assays (ELISA) (Duo-Set ELISA kit; BD Biosciences, San Diego, CA, USA) as per the manufacturer’s instructions. The detection ranges of assays for IL-12p70, IL-10, IL-4 and TNF were 7.8 to 500 pg/ml, and those for the IFN-γ and IL-6 assays were 4.7 to 300 pg/ml. For all the cytokines, absorbance was read at 450 nm using an ELISA plate-reader (Bio-Rad Laboratories, Hercules, CA, USA), and cytokine concentrations were calculated using Bio-Rad microplate manager software. Any values below the detection limit were treated as zero during further analysis.

Levels of inflammatory cytokines TNF-ɑ and IL-6 were assessed using a DuoSet ELISA kit (555268 and 555240, respectively, BD Biosciences, San Diego, USA) according to the manufacturer's instructions. In brief, the whole blood was collected from left ventricle and transferred to EDTA tube (367835, BD Biosciences, San Diego, USA). After centrifugation at 2,000 rpm for 10 min, plasma from blood was prepared and stored in a deep freezer at −80°C until use. 96-well plates were coated with capture antibody in ELISA coating buffer and incubated overnight at 4°C. The plates were then washed with wash buffer and subsequently blocked with assay diluent at RT for 1 h. Diluted standard and plasma samples were added to plates and incubated at RT for 2 h. Detection antibody and streptavidin-conjugated horseradish peroxidase were added to the plates, followed by incubation at RT for 1 h. Tetramethylbenzidine substrate was added and incubated at RT for 0.5 h. The reaction was ended by adding stop buffer. The optical density was measured at 450 nm on an automated ELISA reader (Versa Max, Molecular Devices, CA, USA).