**6. Polymer-based immunohistochemistry**

Desire for IHC detection systems with improved sensitivity led to the development of chain polymer-conjugate technology in the last decade of the former century [69, 70]. Improved sensitivity of this technology is based on using synthetic or natural polymers that increase the capacity for incorporating ligands or enzymes to be coupled to linker antibodies [71–78]. Using this technology, much higher antigen detectability could be obtained in comparison to standard ABC and LSAB methods or in enzyme-antienzyme immune complex techniques (PAP and APAAP) [69, 70]. The chain polymer-conjugate technology normally utilizes a backbone of an inert polymer molecule of dextran [71–73], polypeptides [74], dendrimers [75, 77], or DNA branches [78]. The backbone is able to carry both antibodies and multiple enzymes. Hence, nearly 11 antibodies and up to 40 HRP molecules could be anchor to one 500 KDa dextran molecule [79].

In 1993, a one-step direct polymer immunohistochemical staining method, namely enhanced polymer one-step staining (EPOS) system, was introduced by Bisgaard and Pluzed [80]. In this method, up to 10 monoclonal primary antibodies and 70 enzyme molecules are attached to a dextran backbone with a high molecular weight. This would enable the whole immunohistochemical staining process (from primary antibody to enzyme) to be completed in a single step (**Figure 6a**) [81]. The whole process can be performed in nearly 7 min for frozen sections and to less than 3 h for regularly processed, paraffin-embedded specimens. Hence, when a quick and reproducible IHC-based diagnostic approach is demanded in emergency circumstances, for example, during surgeries, this method should be taken into consideration [82]. However, applicability of this method is restricted to primary antibodies provided by the manufacturer and was not suitable for user supplied primary antibodies.

To overcome this limitation, a polymer-enhanced two-step IHC detection system (EnVision, EV) was introduced in 1995. EV system contains secondary antimouse and antirabbit Ig antibodies and could be applied to localize tissue-bound primary antibodies of mouse and rabbit origins (**Figure 6b**) [83–85]. The EnVision complex is composed of up to 20 secondary antibodies and nearly 100 molecules of peroxidase molecules, which all are directly attached to an activated dextran polymer backbone [86].

EnVision is a user-friendly technique and provides the users a rapid visualization in only 45 min. This method offers a very high sensitivity and does not lead to false-positive reaction due to the endogenous biotin [87]. Although the EV system is a very expensive method, it can be applied with higher dilutions of primary antibodies. Indeed, because endogenous biotin is not a problem anymore, EV permits more efficient heat-induced epitope retrieval (HIER) [69, 88]. The detection systems based on polymers could also be a choice for quick immunostaining of frozen sections when tumor margin and micrometastasis is to be identified. Furthermore, polymer-based detection systems are sensitive enough to be applied as an alternative detection system in western blotting [89] and in chromogenic *in situ* hybridization (CISH) [90].

**Figure 6.** *Polymer-based immunostaining method: (a) EPOS and (b) EnVision.*

Dextran carriers with a high molecular weight, however, appear to compromise the penetrative ability of the detection reagent due to spatial hindrance. Accordingly, the sensitivity of polymer-enhanced systems is profoundly affected by antigen localization. For instance, remarkably low sensitivity has been noticed in nuclear antigens [88, 91]. Indeed, in thick tissue sections, where the antigens are located beneath the surface area, only a part of antigens are amplified. This happens because of the large size of dextran-enzyme complex, which could not disperse into the deeper layers making quantitative results unreliable [87]. Subsequently, EnVision+ was developed, which was a modified version of EV system with higher sensitivity. EnVision+ contains a mixture of dextran polymers with two different secondary antibodies (goat antirabbit and goat antimouse IgG) anchored to it [86, 88, 92]. Nonetheless, EnVision systems were reported to give less sensitivity in case of some antibodies especially those that require proteolytic digestion, which was believed to stem from problems of tissue penetration of the labeled polymer.

Although the application of polymer gives a chance of increasing the number of enzymes coupled to the carrier backbone, it also profoundly increases the size of complex. Therefore, enzyme density per unit surface may not be increased to the degree that would be expected. Hence, it would be a desirable approach to design a compact polymer-enzyme-linker antibody conjugates with optimal number of enzyme molecules. Based on this goal, Shi et al. [91] suggested to use small linear molecules that have a capacity to polymerize with enzymes and linker antibodies in a tightly packed size. The IHC results with this newly designed detection system (Power Vision) showed that it possesses compact size and, compared to conjugates containing polymer linkers, shows higher detection efficiency for antigens located on the cell surface or in the nucleus [91]. Compared to EnVision+, this "second-generation" polymer-based conjugate was found to

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

cal margins of tumor specimens) are needed [95].

**7. Tyramide-based signal amplification**

Adams [99].

**7.1 Biotinylated tyramide signal amplification**

where HRP had previously accumulated (**Figure 7**).

be less expensive and fast and showed better reproducibility and capacity to be standardized [93, 94]. From clinical point of view, these methods are extremely useful when emergency results (for example, assessing the intraoperatively surgi-

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

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

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 avidin-

biotin procedure followed by the tyramine reaction. Background can also be considered a serious problem, particularly with HIER. In this case, more prolonged *Immunohistochemistry - The Ageless Biotechnology*

Dextran carriers with a high molecular weight, however, appear to compromise the penetrative ability of the detection reagent due to spatial hindrance. Accordingly, the sensitivity of polymer-enhanced systems is profoundly affected by antigen localization. For instance, remarkably low sensitivity has been noticed in nuclear antigens [88, 91]. Indeed, in thick tissue sections, where the antigens are located beneath the surface area, only a part of antigens are amplified. This happens because of the large size of dextran-enzyme complex, which could not disperse into the deeper layers making quantitative results unreliable [87]. Subsequently, EnVision+ was developed, which was a modified version of EV system with higher sensitivity. EnVision+ contains a mixture of dextran polymers with two different secondary antibodies (goat antirabbit and goat antimouse IgG) anchored to it [86, 88, 92]. Nonetheless, EnVision systems were reported to give less sensitivity in case of some antibodies especially those that require proteolytic digestion, which was believed to

Although the application of polymer gives a chance of increasing the number of enzymes coupled to the carrier backbone, it also profoundly increases the size of complex. Therefore, enzyme density per unit surface may not be increased to the degree that would be expected. Hence, it would be a desirable approach to design a compact polymer-enzyme-linker antibody conjugates with optimal number of enzyme molecules. Based on this goal, Shi et al. [91] suggested to use small linear molecules that have a capacity to polymerize with enzymes and linker antibodies in a tightly packed size. The IHC results with this newly designed detection system (Power Vision) showed that it possesses compact size and, compared to conjugates containing polymer linkers, shows higher detection efficiency for antigens located on the cell surface or in the nucleus [91]. Compared to EnVision+, this "second-generation" polymer-based conjugate was found to

stem from problems of tissue penetration of the labeled polymer.

*Polymer-based immunostaining method: (a) EPOS and (b) EnVision.*

**12**

**Figure 6.**

be less expensive and fast and showed better reproducibility and capacity to be standardized [93, 94]. From clinical point of view, these methods are extremely useful when emergency results (for example, assessing the intraoperatively surgical margins of tumor specimens) are needed [95].
