Staining enhancement and signal amplification

The method of tyramine amplification is based on the ability of HRP to catalyze the deposition of large amounts of biotinylated tyramine in the vicinity of the antigen-antibody reaction (i.e. the cellular site which contains the peroxidase detection complex). The method is based on earlier biochemical observations on the action of peroxidase upon tyramine and other related compounds (GROSS AJ and SIZER IW, 1959). However, the use of peroxidase to catalyze the deposition of phenols as principle for signal amplification in immunoassays was first reported by MN BOBROW et al. (1989). This signal amplification method was called catalyzed reporter deposition (CARD). The novel method made use of an analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter on the surface in a solid-phase immunoassay; horseradish peroxidase was used as ADRE to catalyze the deposion of biotin labeled phenols. The deposited biotins were then reacted with streptavidinlabeled enzyme resulting in deposition of enzyme. Consequently, the deposition of additional enzyme resulted in an amplification of the signal of the ADRE alone which improved the detection limit of the assay.