Tyramide Signal Amplification TSA Immunocytochemistry

Tyramide signal amplification (TSA) is a powerful amplification method that allows fluorescent labeling to be significantly amplified. TSA is a patented technology from Perkin Elmer that has been licensed to other companies for use in reagents. 

The key to this detection method is the very small compound, tyramine, with two reactive groups (Fig. 7.8). Tyramine contains an amine (-NH2 group), which binds a label (e.g., fluorophore or biotin) on one end and contains a hydroxyl (-OH group) on the other end. When bound to a label via the amine group, the molecule is called tyramide. In the presence of HRP, the inactive tyramide has the OH group, which now binds to a 1° amine on any protein of the tissue. For example, the P antibody, rabbit anti-antigen is bound by the 2° antibody, goat anti-rabbit 

There are three attributes of the TSA procedure that makes the method very powerful. First, the activated tyramide reacts quickly with tyrosine and cannot diffuse far from the HRP-labeled 2° antibody. Second, is that the tyramide is bound to the tissue and is not free to move after the reaction is completed. These advantages are compared to development of many chromogens by HRP where the reaction product is not bound to the tissue and is able to move. Third, there are a wide variety of label compounds that can be attached to tyramine, including both fluorophores and biotin. It is possible to use tyramide bound to biotin and then an avidin HRP, so that a chromogen can be used to localize the 1° antibody. 

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Fig. 7.9 Tyramide signal amplification (TSA) immunocytochemistry. The 1° antibody binds to the antigen and a 2° antibody labeled with HRP binds to the 1° antibody. The labeled tyramide reagent is added with a small amount of the catalyst H2O2. The enzyme HRP converts the inactive labeled tyramide into a reactive labeled tyramide that binds to the amino acid tyrosine found in proteins...

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