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

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

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 constant of avidin binding to biotin (1015 M−<sup>1</sup> ) is nearly 103 –106 times more than the binding affinity of antibody-antigen interaction [22].

The potential of avidin-biotin system to be used in immunoassays was inspired for the first time from a study in 1972 in which it has been shown that avidin could inactivate the biotinylated bacteriophages [23]. Avidin-biotin-based system was used for the first time in an immunological experiment in 1976 when an erythrocyte surface antigen was localized by using biotin-labeled antibody and ferritin-avidin conjugate [24]. In 1979, Guesdon et al. showed that avidin-biotin complex could be effectively used for immunoassays. Using avidin-biotin system, they suggested different related methods for enhancing the specificity and sensitivity of solid-phase immunoassays. They also used avidin-biotin-based immunohistochemistry for localization of intracellular immunoglobulins [25].

The principle of labeled avidin-biotin (LAB) technique is based on sequential interaction of biotin-labeled antibody with tissue antigen and enzyme-labeled avidin with biotinylated primary antibody (**Figure 4a**). In the bridged avidin-biotin (BRAB) technique, however, avidin bridges biotin-labeled primary antibody and biotin-labeled enzyme (**Figure 4b**). BRAB is particularly suitable in cases where intracellular penetration and/or sensitivity of the staining reaction are the major concerns. An indirect approach of BRAB technique (IBRAB) can also be applied for identification of antigens in formalin-fixed paraffin-embedded tissues in which avidin and biotin-labeled peroxidase are added sequentially to the system after primary antibody and biotin-labeled secondary antibody. The superiority of BRAB over LAB method is that there is no need to prepare protein-protein conjugate [20, 25].

Avidin extracted from egg white is a large tetrameric glycoprotein with molecular weight of about 66 KDa. Each subunit (MW 16,400 Da) contains one high-affinity binding site for biotin [26] and one oligosaccharide modification (Asn-linked). Tryptophan and lysine residues in each subunit are believed to be involved in forming the binding pocket with high affinity for biotin molecule [27–29]. Biotin with molecular weight of about 244.31 Da is a small molecule, which has only one binding site specific for avidin. Biotin can be easily conjugated with an antibody or other macromolecules such as fluorochrome and enzymes through other sites [6, 22].

Due to some limitations mentioned below, avidin has been mostly substituted by streptavidin in IHC applications. In this regard, labeled streptavidin-biotin methods (LSAB) are now more popular than LAB methods in both diagnostic and research IHC laboratories [30–34]. The sequence of streptavidin from *Streptomyces avidinii* shows only 30% similarity to avidin, while it has nearly identical secondary, tertiary, and quaternary structure [35]. It is a nonglycosylated protein with a molecular weight of 60 KDa [36]. Avidin is a basic glycoprotein (pI ~ 10.5) that contains nearly 7% carbohydrates, which gives avidin a natural tendency to nonspecifically bind to lectin-like substances found in kidney, liver, brain, and mast cells [7, 36–38]. Avidin can also bind electrostatically to negatively charged tissue elements at physiological pH. Sterptavidin (pI ~ 6.5), however, remains uncharged at neutral pH and does not contain carbohydrate in its structure eliminating its nonspecific binding to tissue lectins. In addition, streptavidin shows less propensity for aggregation [7, 39–41]. Although avidin shows higher binding affinity to free, unconjugated biotin, streptavidin has more tendency to bind biotin-protein conjugates [42]. Indeed, lot to lot variations in binding affinity of biotin and avidin have been reported, which negatively affect sensitivity and reproducibility of the procedure [7].

LAB/LSAB methods offer several advantages for IHC applications. Biological activities of macromolecules (e.g., enzymatic catalysis or antibody binding) are not affected when they are conjugated with biotin. On the other hand, the affinity of avidin/streptavidin to biotin is quite high enough that ensures the biotin-avidin/

**9**

**Figure 4.**

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

with unlabeled primary antibody [47].

*LAB/LSAB (a) and BRAB (b) immunostaining methods.*

biotin, respectively [57–60].

streptavidin complex is not disrupted by manipulations like multiple washing when the complex is immobilized in the tissue sections or by changes in pH and presence of chaotropes [22]. LAB/LSAB techniques considerably improve the sensitivity and efficiency of the immunohistochemical detections and allow researchers to use even more diluted primary antibodies. An immunohistochemical staining that employs a single layer of biotin-labeled monoclonal antibody provides sensitivity equivalent or much greater than PAP methods [43]. The increased sensitivity of avidin-biotin methods stems from larger numbers of biotin molecules that is conjugated to a primary antibody [20, 25, 44]. Due to very high sensitivity, IHC stainings using LAB/ LSAB techniques are rapid [45, 46]. LAB and LSAB technique can also be applied in an indirect manner, where biotinylated secondary antibodies are used in conjunction

The main challenge of LAB/LSAB techniques is the nonspecific (false-positive) staining, which occurs when the tissue of target contains endogenous biotin [6, 7]. Endogenous avidin biotin activity (EABA) or tissue affinity for avidin/ streptavidin is especially common in tissues and cells that contain high amount of biotin, such as placenta, mammary glands, kidney, adrenal cortex, brain, liver, fat, and mast cells [3, 6, 48]. EABA is much highlighted by heat-induced epitope retrieval (HIER) but also develops in tissues subjected to other types of antigen retrieval [49–51]. The level of endogenous biotin activity is especially higher in frozen compared to FFPE tissue sections, which leads to unwanted nonspecific reaction [52, 53]. EABA is typically found in cytoplasm, but it has been reported in the nucleus as well [51, 54–56]. Although paraffin embedding and formalin fixation have been found to significantly decrease the level of endogenous biotin, it is highly recommended to use a biotin blocking step when using avidin/streptavidin-biotin-based detection systems to decrease endogenous biotin activity. Since the commercially available EABA blocking reagents (pure avidin and biotin solutions) are very expensive, many researchers prefer to use homemade blocking reagent containing egg white and 5% powdered milk as sources of avidin and

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

*Immunohistochemistry - The Ageless Biotechnology*

localization of intracellular immunoglobulins [25].

sensitivity and reproducibility of the procedure [7].

through other sites [6, 22].

The potential of avidin-biotin system to be used in immunoassays was inspired for the first time from a study in 1972 in which it has been shown that avidin could inactivate the biotinylated bacteriophages [23]. Avidin-biotin-based system was used for the first time in an immunological experiment in 1976 when an erythrocyte surface antigen was localized by using biotin-labeled antibody and ferritin-avidin conjugate [24]. In 1979, Guesdon et al. showed that avidin-biotin complex could be effectively used for immunoassays. Using avidin-biotin system, they suggested different related methods for enhancing the specificity and sensitivity of solid-phase immunoassays. They also used avidin-biotin-based immunohistochemistry for

The principle of labeled avidin-biotin (LAB) technique is based on sequential interaction of biotin-labeled antibody with tissue antigen and enzyme-labeled avidin with biotinylated primary antibody (**Figure 4a**). In the bridged avidin-biotin (BRAB) technique, however, avidin bridges biotin-labeled primary antibody and biotin-labeled enzyme (**Figure 4b**). BRAB is particularly suitable in cases where intracellular penetration and/or sensitivity of the staining reaction are the major concerns. An indirect approach of BRAB technique (IBRAB) can also be applied for identification of antigens in formalin-fixed paraffin-embedded tissues in which avidin and biotin-labeled peroxidase are added sequentially to the system after primary antibody and biotin-labeled secondary antibody. The superiority of BRAB over LAB

method is that there is no need to prepare protein-protein conjugate [20, 25]. Avidin extracted from egg white is a large tetrameric glycoprotein with molecular weight of about 66 KDa. Each subunit (MW 16,400 Da) contains one high-affinity binding site for biotin [26] and one oligosaccharide modification (Asn-linked). Tryptophan and lysine residues in each subunit are believed to be involved in forming the binding pocket with high affinity for biotin molecule [27–29]. Biotin with molecular weight of about 244.31 Da is a small molecule, which has only one binding site specific for avidin. Biotin can be easily conjugated with an antibody or other macromolecules such as fluorochrome and enzymes

Due to some limitations mentioned below, avidin has been mostly substituted by streptavidin in IHC applications. In this regard, labeled streptavidin-biotin methods (LSAB) are now more popular than LAB methods in both diagnostic and research IHC laboratories [30–34]. The sequence of streptavidin from *Streptomyces avidinii* shows only 30% similarity to avidin, while it has nearly identical secondary, tertiary, and quaternary structure [35]. It is a nonglycosylated protein with a molecular weight of 60 KDa [36]. Avidin is a basic glycoprotein (pI ~ 10.5) that contains nearly 7% carbohydrates, which gives avidin a natural tendency to nonspecifically bind to lectin-like substances found in kidney, liver, brain, and mast cells [7, 36–38]. Avidin can also bind electrostatically to negatively charged tissue elements at physiological pH. Sterptavidin (pI ~ 6.5), however, remains uncharged at neutral pH and does not contain carbohydrate in its structure eliminating its nonspecific binding to tissue lectins. In addition, streptavidin shows less propensity for aggregation [7, 39–41]. Although avidin shows higher binding affinity to free, unconjugated biotin, streptavidin has more tendency to bind biotin-protein conjugates [42]. Indeed, lot to lot variations in binding affinity of biotin and avidin have been reported, which negatively affect

LAB/LSAB methods offer several advantages for IHC applications. Biological activities of macromolecules (e.g., enzymatic catalysis or antibody binding) are not affected when they are conjugated with biotin. On the other hand, the affinity of avidin/streptavidin to biotin is quite high enough that ensures the biotin-avidin/

**8**

**Figure 4.** *LAB/LSAB (a) and BRAB (b) immunostaining methods.*

streptavidin complex is not disrupted by manipulations like multiple washing when the complex is immobilized in the tissue sections or by changes in pH and presence of chaotropes [22]. LAB/LSAB techniques considerably improve the sensitivity and efficiency of the immunohistochemical detections and allow researchers to use even more diluted primary antibodies. An immunohistochemical staining that employs a single layer of biotin-labeled monoclonal antibody provides sensitivity equivalent or much greater than PAP methods [43]. The increased sensitivity of avidin-biotin methods stems from larger numbers of biotin molecules that is conjugated to a primary antibody [20, 25, 44]. Due to very high sensitivity, IHC stainings using LAB/ LSAB techniques are rapid [45, 46]. LAB and LSAB technique can also be applied in an indirect manner, where biotinylated secondary antibodies are used in conjunction with unlabeled primary antibody [47].

The main challenge of LAB/LSAB techniques is the nonspecific (false-positive) staining, which occurs when the tissue of target contains endogenous biotin [6, 7]. Endogenous avidin biotin activity (EABA) or tissue affinity for avidin/ streptavidin is especially common in tissues and cells that contain high amount of biotin, such as placenta, mammary glands, kidney, adrenal cortex, brain, liver, fat, and mast cells [3, 6, 48]. EABA is much highlighted by heat-induced epitope retrieval (HIER) but also develops in tissues subjected to other types of antigen retrieval [49–51]. The level of endogenous biotin activity is especially higher in frozen compared to FFPE tissue sections, which leads to unwanted nonspecific reaction [52, 53]. EABA is typically found in cytoplasm, but it has been reported in the nucleus as well [51, 54–56]. Although paraffin embedding and formalin fixation have been found to significantly decrease the level of endogenous biotin, it is highly recommended to use a biotin blocking step when using avidin/streptavidin-biotin-based detection systems to decrease endogenous biotin activity. Since the commercially available EABA blocking reagents (pure avidin and biotin solutions) are very expensive, many researchers prefer to use homemade blocking reagent containing egg white and 5% powdered milk as sources of avidin and biotin, respectively [57–60].
