Opteia human il 1β enzyme linked immunosorbent assay elisa kit

IL-1β levels were measured using the BD OptEIA^™ Human IL-1β enzyme-linked immunosorbent assay (ELISA) kit (BD-Pharmingen, San Diego, CA). Briefly, a 96-well micro well plate, designed for ELISA (Fisher, Pittsburgh, PA), was coated with a capture antibody for IL-1β diluted in coating buffer. The plate was incubated with the capture antibody overnight at 4 °C. After incubation, the capture antibody was removed by washing the plate three times with wash buffer (PBS and 0.05% Tween-20). Assay diluent (PBS and bovine calf serum) was added to each well (blocking non-specific binding) and incubated at room temperature for 1h. The assay diluent was removed by washing the plate three times, and the cell supernatants and IL-1β standards were added to the coated plated and incubated for 2 h at room temperature. Following this incubation, the plate was thoroughly washed five times and then incubated for 1h with a detection antibody to IL-1β which was conjugated with horseradish peroxidase. The excess detection antibody was removed by washing seven times and a substrate solution was added for 30 min at room temperature to produce a colored product. The incubation with the substrate was ended by addition of acid and the absorbance was measured at 450 nm on a Thermo Labsystems Multiskan MCC/340 plate reader (Fisher Scientific).