Assay set up is similar to a conventional ELISA, using Biotin-streptavidin-HRP, but includes the addition of an amplification reagent I (contains biotinyl-tyramide).

The innovative key substance here is biotinyl-tyramide that is added to the preparaton after streptavidin biotin peroxidase complex. The trick lies in the enzyme HRP using the biotinyl-tyramide as a substrate and catalyzing it from soluble to a highly reactive intermediate that  binds covalently to any protein in the well, e.g. blocking proteins but predominantly in close proximity to the peroxidase. The Result: Additional biotin- binding sites in the area of the relevant antigen/HRP. The amount of reacted biotinyl-tyramide is proportional to the amount of HRP in the well.

After removal of excess biotin-Tyramide from the wells, streptavidin-HRP (Amplification Reagent II) is added, and binds to the additional biotin sites created during the biotin-Tyramide reaction. This amplification step effectively multiplies the quantity of HRP molecules for the final substrate reaction.