Mouse il 17a elisa max

IL-17A ELISA was performed according to the manufacturer’s instructions (Mouse IL-17A ELISA MAX; Biolegend, USA). Nunc MaxiSorp plates (Thermo, AUS) were coated overnight with an IL-17A capture antibody diluted in carbonate coating buffer. The plates were blocked with 1% BSA/PBS for 1 h prior to washing. Splenocyte supernatants (diluted 1:4 in duplicate) and standards were incubated at room temperature for 2 h. The detection antibody was incubated for 1 h prior to avidin–HRP incubation for 30 min. TMB substrate solution was incubated for 20 min, followed by the addition of an acid stop solution. The absorbance (OD) was measured at 450 nm with a correction of 570 nm. IL-17A concentration was determined by plotting the unknown samples on a standard curve to determine IL-17A pg/mL.

Pro-inflammatory cytokines from BAL and plasma were analyzed by an electrochemiluminescence multiplex assay (Meso Scale Discovery, MSD). IL-13 and IL-17 were measured using the kits Mouse IL-13 CytoSet (Thermo Fisher Scientific) and Mouse IL-17A ELISA Max (BioLegend) according to manufacturer’s recommendations.