2.6. Enzyme-Linked Immunosorbent Assay (ELISA) for IL-6, TNF-α, and IL-1β

The IL-6, TNF-α, and IL-1β concentrations in culture supernatants were measured by sandwich ELISA. Monoclonal capture antibodies (4 μg/mL; R&D Systems) were added to 96-well plates (Nunc), and the plates were incubated for 2 hours at room temperature. The plates were incubated with blocking solution consisting of phosphate-buffered saline (PBS) containing 1% BSA and 0.05% Tween 20 for 2 hours at room temperature. The test samples and standard recombinant IL-6, TNF-α, and IL-1β (R&D Systems) were added to the plates, and the plates were incubated overnight at 4°C. The plates were washed four times with PBS containing Tween 20; then, 200 ng/mL of biotinylated detection monoclonal antibodies (R&D Systems) was added, and the plates were incubated for 2 hours at room temperature. The plates were washed, streptavidin-alkaline-phosphatase (diluted 1 : 2000; Sigma-Aldrich) was added, and the reaction was allowed to proceed for 2 hours at room temperature. The plates were washed four times, and 1 mg/mL of p-nitrophenyl phosphate dissolved in diethanolamine (both from Sigma-Aldrich) was added to induce the color reaction, which was stopped by adding 50 μL of 1 N NaOH. The optical density at 405 nm was measured on an automated microplate reader (Bio-Rad, Hercules, CA, USA). Standard curves were drawn by plotting optical density versus the logs of IL-6, TNF-α, and IL-1β concentrations.