Catalyzed signal amplification

Different authors and commercial suppliers have assigned different names to signal amplification using tyramine. For example, tyramine signal amplification (TSA) system and the catalyzed signal amplification (CSA) system are comma-daily available from DuPont NEN Life Science Products, Boston, MA, and DAKO Corporation, Carpinteria, CA, respectively. In addition, the tarns CARD (catalyzed reporter deposition) (Bobrow et al., 1989), TA (tyramide amplification) (Shindler and Roth, 1996), and ImmunoMax (Merz et al., 1995) have been used for the tyramine amplification technique. The use of different names for almost identical procedures has resulted in confusion. To standardize the terminology, the neutral abbreviation, tyramide amplification technique (TAT) should be accepted (Von Wasielewski et al., 1997). 

Figure 2. Highly sensitive immunohistochemical detection method. The catalyzed signal amplification system, which employs fluoresceinyl tyramide (FT-CSA), is a representative enhancement technique that is suitable for pathological research with archival tissue sections. The method consists of two steps, an amplification step by first-step peroxidase-labeled antibody and fluoresceinyltyramide (a), and a visualization step by second-step peroxidase-labeled antibody and chromogen (b), in both of which microscopic observation of immunosignals is possible (inset photos HER4 IHC analysis in A431 epidermoid cell lines).

Hashizume K, Hatanaka Y, Kamihara Y, et al. Automated immunohistochemical staining of formalin-fixed and paraffinembedded tissues using a catalyzed signal amplification method. Appl. Immunohistochem. Mol. Morphol. 2001 9 54-60. 

The sequence of streptavidin-peroxidase and biotinyl-tyraminde can be alternately applied to perform a cycled tyramide amplification procedure. In practicality, however, cycling usually cannot exceed two or three cycles before background staining limits the utility of this approach. Commercial tyramide amplification products are available and include Tyramide Signal Amplification (TSA, DuPont NEN Life Sciences, Boston, MA) and Catalyzed Signal Amplification (CSA). ... 

Use, for example, Vectastain ABC method (Vector Laboratories, Burlingame, CA), or CSA Dakor (catalyzed signal amplification system for mouse primary antibodies) Dako and APAAP mouse or rat primary antibody kits (Dako). 

ABC avidin-biotin complex, AP alkaline phosphatase, ATS all types of sections, CSA catalyzed signal amplification, EPOS enhanced polymer one step (Dako, Glostrup, DK), HIER heath induced epitope retrieval, HRP horseradish peroxidase, lEnz immunoenzymatic, IMF immunofluorescence LSAB labeled streptavidin-biotin, PAP peroxidase-anti-peroxidase... 

Tyramide signal amplification This procedure, designated as a catalyzed reporter deposition (CARD) or tyramide signal amplification (TSA), takes advantage of horseradish peroxidase (HRP) from an HRP-labeled secondary antibody to catalyze in the presence of hydrogen peroxide the oxidation of the phenol moiety of labeled tyramine. On oxidation by HRP, activated tyramine molecules rapidly bind covalently to electron-rich amino acids of proteins immediately surrounding the site of the immunoreaction. This allows an increase in the detection of an antigenic site up to 100-fold compared with the conventional indirect method with no loss in resolution. 

Hunyady, B Krempels, K., Harta, G. Y and Mezey, E. (1996) Immunohis-tochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining. J. Histochem. Cytochem. 44, 1353-1362. 

Compared with radioactive ISH, nonradioactive ISH requires a 10- to 50-fold higher concentration of probes such as oligonucleotides. However, signal amplification is decreased by increasing probe concentration. Therefore, since nonradioactive probes have limited sensitivity, especially when applied to low-abundance mRNAs, a technique is required for signal amplification. One such technique consists of an optimized protocol for rapid signal amplification based on catalyzed reporter deposition (CARD) that increases the sensitivity of nonradioactive mRNA ISH on the formaldehyde-fixed and paraffin-embedded tissues (Speel et al., 1998). This technique facilitates the detection of low-copy mRNAs by ISH (Yang et al., 1999). 

Bobrow, M. N., Harris, T. D., Shaughnessy, K. J., and Litt, G. J. 1989. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods 725 279-285. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Bobrow MN, Harris TD, Shaughnessy KJ, et al. Catalyzed reporter deposition A novel method of signal amplification Application to immunoassays. J Immunol Methods. 1989 125 279. 

Bobrow MN, Litt GJ, Shaughnessy KJ, Mayer PC, Conlon J (1992) The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats. J Immunol Meth 150 145-149... 

Hundreds of successful examples of these approaches have already been published. However, there are a few disadvantages in the use of fluorescent proteins a reporters when compared to enzymatic reporter systems. The main disadvantage is the absence of signal amplification. Whereas fluorescent proteins are limited to a single chromophore for each protein, a single copy of 13-galactosidase, luciferase or 13-lactamase will catalyze the turnover of... 

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