ABC with TSA

ABC with TSA offers the largest amount of label amplification and therefore the best detection sensitivity. Combining the two high-detection-sensitivity methods into one procedure when the antigen is present in the tissue at low number of copies or when there are very high dilutions of L antibodies (e.g., 1 500,000). In both cases, the sensitivity of the detection must be maximized. 

The procedure begins with the standard ABC protocol, where biotin-labeled 2° antibody is bound with the avidin-biotin complex (ABC). This adds a significant number of HRP molecules for each L antibody. To start the TSA method, incubate tissue with biotin-labeled tyramide with H2O2 the HRP enzymes generate activated tyramide (Fig. 7.10). The activated tyramide then binds to the available tyrosine amino acids on cellular proteins near the antigen, on the antibodies, and even on 

Two concerns with the ABC with TSA method are related to the ultra high dilutions of 1° antibody and the incomplete labeling of all the antigens within the cell. 

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Fig. 7.11 ABC with TSA detection with fluorescence. With the biotin tyranude generated in the ABC and TSA (Fig. 7.10), detection is with an avidin with fluorescent label. The fifth incubation will give the fluorescent labeling...

Fig. 7.10 ABC and TSA. To obtain the largest amount of signal for a single 1° antibody combine ABC and TSA methods. After the 1° antibody binds the antigen and the 2° antibody labeled with biotin binds to the 1° antibody. The third incubation is with the ABC reagent that labels with an HRP. In a fourth incubation, HRP activation of biotin-labeled tyramide near the antigen, adds numerous biotins to the tissue near the antigen. The labeling is completed with fluorescence (Fig. 7.11) or enzymes (Fig. 7.12)...

FIGURE 11.12 TSA method using the conventional ABC method, together with pretreatment procedures and optimal antigen retrieval. Source www.hmds.org.uk/ histology.html... 

As the TSA-ABC protocol dramatically improves the signal intensity by the peroxidase-catalyzed deposition of biotinylated tyramide, blocking of endogenous peroxidase is required. To ensure quenching of the residual peroxidase, the use of a higher concentration and duration of treatment with H202 is recommended. 

Originally described several years ago for use in immunoassay systems (36), biotinylated tyramine amplification has recently been adapted for immuno-cytochemical use (37). The system is based on the ABC method. H2O2 is catalysed to H2O and oxygen-free radicals by HRP as usual. This then reacts with biotinylated tyramine to produce activated biotinylated tyramine. which binds to nearby proteins in a highly localized manner. The high concentrations of biotin thus produced can then be detected by the application of one additional layer of avidin-reporter conjugate. The system has been used to demonstrate antigens previously only detectable in frozen sections, and to increase primaiy antibody dilution. Several manufacturers produce kits that are based on the system (CSA from Dako Ltd. and TSA from NEN life Science Products), or home-made biotinylated tyramine can be produced. 

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