Analyte-specific antibody (capture antibody) is bound to a user-provided polystyrene microplate. Unbound capture antibody is washed away. Plates are blocked and washed.
Samples or standards are added and any analyte present is bound by the immobilized antibody. Unbound materials are washed away.
Streptavidin-Horseradish Peroxidase (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.