Mouse albumin

ELISA kits for mouse IgA and IgM (eBioscience, Frankfurt am Main, Germany), mouse IL-6 and TNF-α (BioLegend, London, United Kingdom), mouse C5a (R&D Systems, Minneapolis, MN) and mouse albumin (Bethyl Laboratories Inc., Montogomery, TX) were used according to manufacturer’s instructions. For SIgA-detection Nunc MaxiSorp plates were coated overnight with monoclonal rat anti-mouse IgA (SouthernBiotech, Birmingham, 2 µg/mL). Plates were washed, blocked and incubated with serial dilutions of BALF samples and incubated with secondary antibody (polyclonal goat anti-mouse pIgR; R&D Systems, 1 µg/mL) followed by polyclonal donkey anti-goat-HRP (Jackson ImmunoResearch, West Grove, PA, 0.16 µg/mL) and TMB substrate. Plates were read at 450 and 570 nm. Due to the lack of a SIgA protein standard, relative quantification was performed. Results are expressed as the OD at 450 nm (wavelength- and blank-corrected) at indicated dilutions.