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9 Enhanced Tyramide Signal Amplification Immunohistochemical Detection

Tyramide signal amplification (TSA) is a sensitive immunodetection technique based on the peroxidase catalyzed deposition of labeled tyramide molecules. TSA in both direct (fluorophore conjugated) and indirect (biotin or DNP conjugated) formats has been used to amplify signals in a variety of applications including immunoassays, Western blotting, in situ hybridizations, and immunocytochemistry. The increased sensitivity of TSA for immunohistochemistry typically permits primary antisera to be used at concentrations ten-to one hundred fold less than that used when fluorophore-conjugated secondary antisera are employed. In an attempt to increase the sensitivity of TSA immunodetection, we discovered a group of novel substrate compositions which demonstrated increased horseradish peroxidase (HRP) catalytic activity and tyramide deposition. In testing these substrates in immunohistochemical applications, we found a marked enhancement in sensitivity of both direct and indirect TSA detection which was further increased by optimization of the HRP reaction buffer conditions. For example, application of the enhanced TSA reaction conditions to direct TSA detection of microtubule-associated protein-two immunohistochemical detection in mouse brain sections permitted an one-thousand fold reduction in primary antibody concentration compared to conventional immunodetection techniques and an one-hundred fold reduction compared to standard TSA detection. Similarly, the enhanced TSA formation significantly improved the sensitivity of indirect TSA immunohistochemical detection, although to a lesser degree compared to the direct format. This improved TSA immunodetection paradigm provides heretofore unobtainable non-radioactive antigen detection limits and should prove useful for both immunohistochemical and in situ hybridization applications.

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