Enzyme-linked immunosorbent assay

The wells of high-binding 96-well plates were coated with anti-mouse Rae-1 polyclonal antibody (ThermoFisher) overnight at 4 °C, washed and blocked using an ELISA Deluxe Set (BioLegend). The supernatant collected from CT26^Rae1 cells and positive and negative controls were added to the antibody-coated wells for incubation for 2 hours at room temperature, followed by incubation with biotin-labeled anti–Rae-1 monoclonal antibody [33], avidin-horseradish peroxidase solution, and substrate solution from the ELISA Deluxe Set. The reaction was stopped after 20 minutes, and the absorbance was read at 450 nm.