A microplate pre-coated with capture antibody is provided. Samples or standards are added and any analyte present is bound by the immobilized antibody. Unbound materials are washed away.
A second HRP-labeled antibody (detection antibody) is added and binds to the captured analyte. Unbound detection antibody is washed away.
Enhanced Luminol substrate is added and light is produced in proportion to the amount of analyte present in the sample. A microplate luminometer is used to measure the intensity of the light emitted.