Biotinylated anti areg antibody

Human lung H292 cells were plated and stimulated as indicated in respective figures in 24-well plates at 1.5 × 105 cells per well. 300–500 μl of cell supernatant was collected and frozen at −20 °C until further use. Mouse BAL samples were used for mouse Areg ELISA. For Sandwich ELISA Nunc Maxisorp 96-well plates (Thermo Fisher Scientific) were coated with capture human Areg antibody (R&D systems) at 5 μg/ml or mouse Areg antibody (R&D systems) at 0.5 μg/ml and incubated O/N at 4 °C. Plates were washed with PBS-T and blocked with 1 % BSA for 1hr at RT. Human Areg was detected with biotinylated anti-Areg antibody (R&D systems). Mouse Areg was detected with biotinylated anti-Areg antibody (R&D systems). Antibody binding was detected with streptavidin-HRP (BD Biosciences) and developed with TMB substrate set (BD Biosciences). Recombinant human (GeneScript) and mouse Areg (BioLegend) were used as standards.