Anti human il 8 antibody

IL-6, IL-8 and TNFα were measured in the acellular bronchial lavage fluid using a standard sandwich ELISA as described previously (Wyatt et al., 2010 (link)). For IL-6 and IL-8 measurements, microtiter plates were coated with the appropriate capture antibody in Voller’s carbonate buffer (pH 9.6) overnight at 4°C. The anti-human IL-8 antibody (R&D Systems, Minneapolis, MN) was diluted 1:500; IL-6 antibody (R&D Systems) was diluted 1:1000. The plates were washed and recombinant IL-6 or IL-8 standards were applied along with 200µl of samples in duplicate for 2 hours. The plates were washed and the “bridge” antibody was applied, either (rabbit) anti-human IL-8 antibody diluted 1:500 (Rockland, Gilberstville, PA) or IL-6 antibody (R&D Systems) diluted 1:1000 for 1 hour. The plates were washed and then developed using a peroxidase substrate consisting of 10 ng/ml orthophenylenediamine (Sigma-Aldrich), and 0.003% H₂O₂ in distilled water. The TNFα measurements were made in the same way with the following exceptions: The capture antibody was anti-human TNFα (2 µg/mL), the secondary “bridge” antibody was biotinylated (rabbit) anti-human TNF-α (200 ng/mL). Detection was performed using steptavidin-HRP (1:200, R&D Systems). Total protein levels in the fluid was also measured using the Bradford assay (BioRad, Hercules, CA).