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 biotinylated antibody (detection antibody) is added and binds to the captured analyte. Unbound detection antibody is washed away.
Streptavidin-HRP is used to bind to the detection antibody. Unbound streptavidin-HRP is washed away.
Tetramethylbenzidine (TMB) substrate solution is added to the wells and a blue color develops in proportion to the amount of analyte present in the sample. Color development is stopped turning the color in the wells to yellow. The absorbance of the color at 450 nm is measured.