**5.2 Avidin-biotin complex (ABC) method**

For signal amplification, another biotin-based IHC detection method was developed, namely avidin-biotin complex (ABC) method, in which a preformed avidin-biotin-peroxidase complex is used as the detection layer [20, 61]. This technique induces three different layers; an unconjugated primary antibody, a biotinylated secondary antibody, and finally a large complex of enzyme-labeled biotin and avidin, which is attached to the biotin molecules conjugated to the secondary antibodies (**Figure 5**). Two biotins from adjacent biotinylated enzyme molecules can be joined via an avidin molecule [62]. Four biotin binding site of avidin molecule could result in formation of lattice complexes in which avidins are attached together by biotinylated enzyme molecules creating a large complex, which is attached to the biotinylated secondary antibody [20, 63]. In normal circumstances, not all the four avidin's capacity for biotin are taken up by biotinylated enzyme. This allows the complexes to attach to biotin of primary or secondary antibodies [64].

Before the advent of biotin-avidin-based methods, the PAP method was considered the most sensitive detection technique. The ABC method was then found to be nearly 40 times more sensitive compared to PAP method [65, 66].

By applying biotinylated primary antibodies, the ABC protocol can be shortened to a two-step method [3]. It is reported that application of biotin-labeled primary antibody in the two-step ABC method creates an equal sensitivity to the unconjugated antibody in the three-step ABC method. This finding proposes that biotinylation does not impair antibody activity and that application of a secondary antibody to intensify the reaction would not be necessary, if a suitable biotin-labeled primary antibody is used [43].

Although formation of lattice complexes of avidin and biotinylated enzyme seems to increase the sensitivity of ABC method compared to LAB/LSAB, it was found that

**11**

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

**6. Polymer-based immunohistochemistry**

to one 500 KDa dextran molecule [79].

primary antibodies.

polymer backbone [86].

chromogenic *in situ* hybridization (CISH) [90].

the sensitivity of ABC method is 5–10 times less than the LSAB method [67]. This disadvantage of ABC method is due to the large size of lattice complexes that hinder their penetration into the cells. Indeed, as with LAB/LSAB methods, the background in ABC method cannot be removed due to the irreversibility of the avidin-biotin reaction [21, 68]. As with LAB/LSAB methods, tissue endogenous biotin is one of the concerns in ABC-based IHC staining methods that results in nonspecific staining.

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

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

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

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

**Figure 5.** *Avidin/streptavidin immunostaining method.*

*Immunohistochemistry - The Ageless Biotechnology*

**5.2 Avidin-biotin complex (ABC) method**

antibodies [64].

antibody is used [43].

For signal amplification, another biotin-based IHC detection method was developed, namely avidin-biotin complex (ABC) method, in which a preformed avidin-biotin-peroxidase complex is used as the detection layer [20, 61]. This technique induces three different layers; an unconjugated primary antibody, a biotinylated secondary antibody, and finally a large complex of enzyme-labeled biotin and avidin, which is attached to the biotin molecules conjugated to the secondary antibodies (**Figure 5**). Two biotins from adjacent biotinylated enzyme molecules can be joined via an avidin molecule [62]. Four biotin binding site of avidin molecule could result in formation of lattice complexes in which avidins are attached together by biotinylated enzyme molecules creating a large complex, which is attached to the biotinylated secondary antibody [20, 63]. In normal circumstances, not all the four avidin's capacity for biotin are taken up by biotinylated enzyme. This allows the complexes to attach to biotin of primary or secondary

Before the advent of biotin-avidin-based methods, the PAP method was considered the most sensitive detection technique. The ABC method was then found to be

By applying biotinylated primary antibodies, the ABC protocol can be shortened to a two-step method [3]. It is reported that application of biotin-labeled primary antibody in the two-step ABC method creates an equal sensitivity to the unconjugated antibody in the three-step ABC method. This finding proposes that biotinylation does not impair antibody activity and that application of a secondary antibody to intensify the reaction would not be necessary, if a suitable biotin-labeled primary

Although formation of lattice complexes of avidin and biotinylated enzyme seems to increase the sensitivity of ABC method compared to LAB/LSAB, it was found that

nearly 40 times more sensitive compared to PAP method [65, 66].

**10**

**Figure 5.**

*Avidin/streptavidin immunostaining method.*

the sensitivity of ABC method is 5–10 times less than the LSAB method [67]. This disadvantage of ABC method is due to the large size of lattice complexes that hinder their penetration into the cells. Indeed, as with LAB/LSAB methods, the background in ABC method cannot be removed due to the irreversibility of the avidin-biotin reaction [21, 68]. As with LAB/LSAB methods, tissue endogenous biotin is one of the concerns in ABC-based IHC staining methods that results in nonspecific staining.
