**4. Bridge methods**

Early conjugation protocols were not efficient and did not label all antibodies leaving a fraction of antibodies unlabeled. These unlabeled antibodies were able to compete with labeled antibodies for binding to the cognate antigen and reduced the efficiency of detection. To overcome this problem, new approaches were invented that eliminated the need for chemical conjugation of antibodies. In these approaches, antigen specificity of antibodies is employed to couple antibodies to the enzymes. Taking advantage of antigen specificity of antibodies, antiperoxidase or antialkaline phosphatase antibodies are easily coupled with peroxidase or alkaline phosphatase after incubation with these enzymes without need for any chemical modifications of the antibody. These preformed soluble enzyme-antienzyme immune complexes are then used as the third layer reporter antibody for detection of the antigen-bound primary antibody in tissue section. Taking advantage of the bivalent properties of IgG binding, a second-step antibody with binding specificity to primary antibody and tertiary antienzyme antibody complexed with the enzyme bridges two layers (**Figure 3a**). The bridge antibody is usually used in excess, so that one of its two identical binding sites interacts with enzyme-coupled tertiary antibody, while the other site interacts with primary antibody. The tertiary antienzyme antibody has the same animal species of origin as the primary antibody. The bridge methods are collectively called as soluble enzyme-antienzyme methods [5, 8, 15–17].

The classical immunoenzyme bridge method [18] was rapidly replaced with an improved version in which peroxidase-antiperoxidase complex (PAP, MW: 400–430 KDa) contained three peroxidase molecules and two antiperoxidase antibodies (**Figure 3b**) [15]. In this system, antibodies against alkaline phosphatase can also be employed to form alkaline phosphatase-antialkaline phosphatase complexes (APAAP, MW: approximately 560 KDa) [17]. In contrast to PAP complexes, APAAP complexes include two molecules of alkaline phosphatase and only one antibody. The APAAP method is usually used as an alternative to PAP technique when high levels of endogenous peroxidase in such tissues as bone marrow aspirate specimens, spleen and peripheral blood, interfere with the staining or when double labeling approaches are desired [19].

**7**

**Figure 3.**

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

able for cell and cryosection IHC [14].

*Two- (a) and three-step (b) bridge immunostaining methods.*

**5. Biotin-avidin/streptavidin-based methods**

constant of avidin binding to biotin (1015 M−<sup>1</sup>

**5.1 Labeled avidin/streptavidin-biotin (LAB/LSAB)**

binding affinity of antibody-antigen interaction [22].

Soluble enzyme-antienzyme methods offer several advantages over direct and indirect detection methods. The drawback of chemical conjugation process, which could potentially lead to impairment of antibody activity, is entirely avoided in enzyme-antienzyme methods. Due to a greater number of enzyme molecules localized per antigenic site, enzyme-antienzyme process shows the higher sensitivity compared to previously described methods. It is reported that PAP method exhibits nearly 100- to 1000-fold higher sensitivity than two-step indirect method [7]. Although multilayering of detection antibodies could potentially increase the risk for nonspecific interaction with the tissue antigens, PAP and APAAP methods that offer triple level detection are among the exceptions. These methods are suit-

In comparison to two-step indirect methods, PAP and APAAP are more timeconsuming. Indeed, these methods may not have sensitivity enough required for use in formalin-fixed paraffin-embedded (FFPE) preparations, especially when used in combination with monoclonal antibodies [14]. Although PAP and APAAP methods have been known as the highly sensitive, reliable, and popular techniques in pathology laboratories for a long time, they have gradually been replaced by more

Labeled avidin/streptavidin-biotin (LAB/LSAB) are among very sensitive IHC detection methods, which take advantage of high-affinity binding of avidin/streptavidin to a water-soluble vitamin, biotin (vitamin H or B7) [5, 20–22]. The affinity

) is nearly 103

–106

times more than the

improved methods such as streptavidin-biotin- and polymer-based systems.

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

**Figure 3.**

*Immunohistochemistry - The Ageless Biotechnology*

and clinical settings.

**4. Bridge methods**

fluorophore could be a right choice [3]. Previously mentioned advantages of indirect detection systems eventually led to its widespread applications in research

Despite advantages mentioned above, indirect immunostaining methods suffer from some shortcomings. First, additional controls and blocking steps are inevitable when using secondary antibodies. Indeed, there is possibility of nonspecific staining that happens when the secondary antibody interacts with unwanted tissue targets. If nonspecific staining is noticed, blocking reagents have to be used to treat the tissue sections that could be time-consuming and cause additional costs to IHC experiment [6, 13]. The blocking agent should contain nonimmune antibody fraction from the same species in which secondary antibody has been produced. This results in competitive blocking of the nonspecific binding sites for secondary antibody in the target tissue by the unlabeled antibodies from the same species. The addition of further layers beyond their use in the two-step indirect method for increasing the sensitivity of detection can be problematic as addition of every new species of antibody considerably increases the risk of nonspecific interactions and background staining [14]. Desire for more sensitive detection systems triggered researchers to develop next-generation detection systems, especially for those antigenic markers, which are not expressed in physiological condition, and any level

of their upregulation would be interpreted as a pathological condition.

Early conjugation protocols were not efficient and did not label all antibodies leaving a fraction of antibodies unlabeled. These unlabeled antibodies were able to compete with labeled antibodies for binding to the cognate antigen and reduced the efficiency of detection. To overcome this problem, new approaches were invented that eliminated the need for chemical conjugation of antibodies. In these approaches, antigen specificity of antibodies is employed to couple antibodies to the enzymes. Taking advantage of antigen specificity of antibodies, antiperoxidase or antialkaline phosphatase antibodies are easily coupled with peroxidase or alkaline phosphatase after incubation with these enzymes without need for any chemical modifications of the antibody. These preformed soluble enzyme-antienzyme immune complexes are then used as the third layer reporter antibody for detection of the antigen-bound primary antibody in tissue section. Taking advantage of the bivalent properties of IgG binding, a second-step antibody with binding specificity to primary antibody and tertiary antienzyme antibody complexed with the enzyme bridges two layers (**Figure 3a**). The bridge antibody is usually used in excess, so that one of its two identical binding sites interacts with enzyme-coupled tertiary antibody, while the other site interacts with primary antibody. The tertiary antienzyme antibody has the same animal species of origin as the primary antibody. The bridge methods are collectively called as soluble enzyme-antienzyme methods [5, 8, 15–17]. The classical immunoenzyme bridge method [18] was rapidly replaced with an improved version in which peroxidase-antiperoxidase complex (PAP, MW: 400–430 KDa) contained three peroxidase molecules and two antiperoxidase antibodies (**Figure 3b**) [15]. In this system, antibodies against alkaline phosphatase can also be employed to form alkaline phosphatase-antialkaline phosphatase complexes (APAAP, MW: approximately 560 KDa) [17]. In contrast to PAP complexes, APAAP complexes include two molecules of alkaline phosphatase and only one antibody. The APAAP method is usually used as an alternative to PAP technique when high levels of endogenous peroxidase in such tissues as bone marrow aspirate specimens, spleen and peripheral blood, interfere with the staining or when double labeling approaches are

**6**

desired [19].

*Two- (a) and three-step (b) bridge immunostaining methods.*

Soluble enzyme-antienzyme methods offer several advantages over direct and indirect detection methods. The drawback of chemical conjugation process, which could potentially lead to impairment of antibody activity, is entirely avoided in enzyme-antienzyme methods. Due to a greater number of enzyme molecules localized per antigenic site, enzyme-antienzyme process shows the higher sensitivity compared to previously described methods. It is reported that PAP method exhibits nearly 100- to 1000-fold higher sensitivity than two-step indirect method [7]. Although multilayering of detection antibodies could potentially increase the risk for nonspecific interaction with the tissue antigens, PAP and APAAP methods that offer triple level detection are among the exceptions. These methods are suitable for cell and cryosection IHC [14].

In comparison to two-step indirect methods, PAP and APAAP are more timeconsuming. Indeed, these methods may not have sensitivity enough required for use in formalin-fixed paraffin-embedded (FFPE) preparations, especially when used in combination with monoclonal antibodies [14]. Although PAP and APAAP methods have been known as the highly sensitive, reliable, and popular techniques in pathology laboratories for a long time, they have gradually been replaced by more improved methods such as streptavidin-biotin- and polymer-based systems.
