Procartaplex high sensitivity assay

Concentrations of IL-1β in EDTA plasma samples obtained from patients with COPD and healthy individuals were measured using a ProcartaPlex High Sensitivity Assay, with a corresponding IL-1β bead set (Thermo Fisher Scientific, Waltman, MA, USA), according to the manufacturer’s recommendations. Briefly, 50 μL of antibody-coated magnetic beads were added per well into a 96-well plate and washed. Afterwards, 25 μL of samples or standards were added to a 25 μL universal assay buffer, and the plate was incubated for 30 min at room temperature (RT) and overnight at 4°C, with shaking. After the washing steps, 25 μL of detection antibodies were added to the wells and the plate was incubated for 30 min at RT, with shaking. After the washing, 50 μL of a streptavidin-phycoerythrin conjugate was added to the wells. After the incubation and washing steps, 50 μL of amplification reagent 1 was added to the wells, and the plate was incubated for 30 min at RT, with shaking. Then, amplification reagent 2 (50 μL) was added to the wells, and, following the incubation and washing steps, the beads were resuspended in 120 μL of reading buffer and analyzed by a Luminex 200 instrument (Luminex Corporation, Austin, TX, USA). The concentration of IL-1β was determined by interpolation from a standard curve using the xPONENT software package (Luminex Corporation, Austin, TX, USA).