Rabbit anti human il 8 antibody

IL-8 protein levels in media were quantified using quantitative sandwich ELISA. 96-well round-bottom microtiter plates (Costa, Cambridge, USA) were coated with 200 mL/well of rabbit-anti-human IL-8 antibody (R&D, Minnesota, USA) diluted 1:2,000 in Voller’s buffer for 24 h at 4 °C. After three times washing in PBS-Tween, undiluted media and serial dilutions of standard human recombinant RIL-8 (Sigma, Missouri, USA) were incubated at room temperature for 90 min. Plates were rinsed 3 times with PBS-Tween followed by the addition of rabbit-anti-human IL-8 antibody (R&D, Minnesota, USA) diluted 1:2,000 in the washing solution. After 1 h incubation, dishes were washed 3 times, and peroxidase conjugated goat-anti-rabbit (Sigma, Missouri, USA) was added at a 1:2,000 dilution for 1 h incubation. The plates were then washed again, and orthophenyldiamine (Sigma, Missouri, USA) was dissolved in methanol (1 mg/mL) and diluted (10 ng/mL) in distilled water containing 0.01% H2O2 was added. The next reaction was stopped with 50 µL of 2 mol/L sulfuric acid, and plates were read at 492 nm at HTS 7000 Plus Bio Assay Reader (Perkin-Elmer, CT, USA).