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 Alkaline Phosphatase (AP)-labeled antibody (detection antibody) is added and binds to the captured analyte. Unbound detection antibody is washed away.
NADPH substrate solution is added and a rose color develops. Plates are NOT washed.
Amplifier solution is added and the rose color deepens to a red color in proportion to the amount of analyte present in the sample. Stop solution is added (color remains red) and the absorbance of the color at 490 nm is measured.