Detection of IL6 and IL8 was performed by enzyme-linked immunosorbent assay (ELISA), as previously described [21 (link)], using the sandwich method. ELISA plate coating was done using mouse monoclonal anti-human IL8 (clone G265-5, BD Biosciences, San Jose, CA, USA) and IL6 (clone 6708 R&D Systems, Inc.). For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.
BAL fluids were retrieved before and 48 h after SBP-Ag, concentrated 20-fold and IL1α and IL1β concentrations were determined using a DuoSet Development kit (DY-200 and DY-201, respectively, R&D Systems, Inc.), while EDN concentrations were determined using the sandwich ELISA kit (7630, MBL International Corp., Des Plaines, IL, USA). The IL1α and IL1β DuoSet Development ELISA kits and EDN ELISA kit had sensitivities of 1 pg/mL and 1 ng/mL, respectively.
BAL fluids were retrieved before and 48 h after SBP-Ag, concentrated 20-fold and IL1α and IL1β concentrations were determined using a DuoSet Development kit (DY-200 and DY-201, respectively, R&D Systems, Inc.), while EDN concentrations were determined using the sandwich ELISA kit (7630, MBL International Corp., Des Plaines, IL, USA). The IL1α and IL1β DuoSet Development ELISA kits and EDN ELISA kit had sensitivities of 1 pg/mL and 1 ng/mL, respectively.