**7.1 Biotinylated tyramide signal amplification**

In 1989, a novel signal amplification method for immunoassays was introduced by Bobrow et al. called catalyzed reporter deposition (CARD). The CARD was first used in western blots and immunodots [96–98] and was then adapted for IHC by Adams [99].

The signal amplification in this system is based on an analyte-dependent reporter enzyme (ADRE), which catalyzes the deposition of additional reporter molecules. The first step of this system relies on the same principle as LAB/LSAB detection system. Accordingly, primary antibody is first added to the tissue section followed by biotinylated secondary antibody and either HRP-labeled streptavidin (in tyramine signal amplification (TSA)) or streptavidin-biotin-HRP complex (in catalyzed signal amplification (CSA)). The amplification process happens when the peroxidase enzyme (ADRE) oxidizes the phenolic components to produce extremely unstable and reactive intermediate radicals, which are then bound to a tissue section [96, 100]. Tyramine, a biogen amine derived from aromatic amino acid tyrosine, is a substrate commonly used in this technique. It contains an amine at one end and a phenol at another end, which is used by peroxidase enzyme. The amine group is employed to conjugate the molecule with biotin or any other target molecules via an amide bound [101]. In the presence of HRP and H2O2, biotinylated tyramine is oxidized and resulting highly reactive radicals will react with electron-rich aromatic components, such as tyrosinerich moieties of proteins in the vicinity of the HRP binding sites in tissues. This binding occurs very rapidly within 10 min. Due to a very short half-life of tyramide radicals, they are deposited at the same location where they are generated [102]. This reaction is then followed by incubation of the tissues with streptavidin-peroxidase complex. This complex is attached to the biotin sites of the tyramine, which are remained free. This reaction is restricted to the sites of primary antibody binding site where HRP had previously accumulated (**Figure 7**).

Because of the high sensitivity of this method, biotinylated tyramide amplification enabled many antigens to be traced, which had previously been unreactive in formalin-fixed paraffin-embedded tissues [101]. In comparison to the avidinbiotin-based methods, biotinylated tyramide signal amplification exhibits 5- to 10-fold more sensitivity. Some researchers believed in even more sensitivity [103]. It was reported by Sanno et al. that staining of pituitary hormones with CSA showed nearly 100-fold higher sensitivity compared to standard ABC method [104]. It is recommended to use this method when (1) antigen expression in target tissue is extremely low or the amount of antibody available is limited and (2) primary antibodies possess low affinity or are not compatible with paraffin-embedded tissue sections [104, 105]. Repeating the biotinyl-tyramide reaction can further increase the signal intensity. However, this circuit is restricted to only two or three rounds before the background noise becomes an issue [106]. CSA and/or TSA methods are found to be cheaper than EnVision system but with the same effectiveness [86].

These methods, however, are laborious because they involve an initial avidinbiotin procedure followed by the tyramine reaction. Background can also be considered a serious problem, particularly with HIER. In this case, more prolonged

treatment of tissues to quench endogenous peroxidase or endogenous avidin-biotin activities (EABA) is usually necessary [105, 107–109]. Although TSA/CSA detection methods have resulted in satisfactory results in terms of significantly increased sensitivity in IHC and *in situ* hybridization (ISH), they are not widely employed in diagnostic pathology. The reasons include: additional steps that make the method more time-consuming, nonspecific background staining, and that optimal AR treatment with existing methods may achieve equivalent results and that secondgeneration polymer-based methods are simpler and equally sensitive [14, 110, 111].

#### **7.2 Biotin-free TSA/CSA**

In an attempt to reduce the problems associated with endogenous biotin in conventional tyramide signal amplification, a biotin-free system, fluorescyl-tyramide amplification system (FT-CSA or CSAII), was introduced. Rather than biotinyltyramide, this system uses fluorescyl-tyramide and does not contain avidin/biotin reagents avoiding the problem associated with endogenous biotin. In this method, addition of primary antibody is followed by a peroxidase-labeled secondary antibody. Peroxidase enzyme is responsible to catalyze the transformation and deposition of fluorescyl-tyramide in the tissue section. When the reaction terminates, it could be inspected by fluorescence microscopy. The produced signals could even be converted to a colorimetric reaction by using peroxidase-conjugated antifluorescein antibody and a diaminobenzidine-hydrogen peroxide substrate.

This method is highly sensitive enabling researchers to detect and localize antigens with low expression level and to use primary antibodies with very low affinities [105, 106]. Alternative reporter includes dinitrophenol, which also results in marked reduction of background from endogenous biotin. Absence of nonspecific staining is due to no endogenous tissue distribution of dinitrophenol [14].

In the latest improvement of the biotin-free CSA method, fluorescein is conserved in the substrate, while the tyramine is substituted with ferulic acid, which is a much better peroxidase substrate and increases signal-to-noise ratio. In this system, the incubation time in each step can be significantly reduced, making it possible to stain a tissue in less than 1 h [112].

**15**

**Figure 8.**

*Detection Systems in Immunohistochemistry DOI: http://dx.doi.org/10.5772/intechopen.82072*

**8. Rolling circle amplification**

Rolling circle amplification (RCA) reaction was first developed for the purpose of nucleic acid detection [106], but it was then adapted for amplification of signals from antibodies bound to antigens [113–118]. RCA is an enzymatic process in which a short DNA or RNA primer is amplified using a circular DNA template and special DNA or RNA polymerases to form a long single-stranded DNA or RNA [119, 120]. The end product of RCA is a long continuous sequence of DNA containing several tandem repeats complementary to the circular template. Unlike PCR, RCA could be performed at a constant temperature (room temperature to 37°C). A RCA reaction contains five different components: (i) a short DNA or RNA primer, (ii) a polymerase enzyme (e.g., Phi29 DNA polymerase for DNA, and T7 RNA polymerase for RNA), (iii) a suitable buffer compatible with polymerase enzyme, (iv) a circular

DNA template, and (v) deoxy nucleotide triphosphates (dNTPs) [121].

RCA reaction has three different steps: (1) the circular DNA template with typically ~15–200 nt in length is synthesized through the intramolecular ligation of phosphate and hydroxyl end groups of a linear probe with the use of the target DNA or RNA as a ligation template [121–123], (2) the polymerase enzyme continuously adds dNTPs to a circular template-annealed primer to form a long ssDNA with tens to hundreds of tandem repeats, and (3) the RCA end products could be detected and even monitored by different signal readout methods (**Figure 8**) [121]. Different methods are available to visualize and also analyze the RCA process including (a) labeling the RCA products directly during the amplification process by using

*Rolling circle amplification immunostaining method. (1) Immunoconjugate bound to target antigen. (2) RCA primer hybridized to circle template (3) Synthesis of new DNA strand by DNA polymerase (4) Detection of* 

*amplified DNA by enzyme-labeled probe at the site of bound antibody.*
