Avidin-Biotin Immunocytochemistry

In immunocytochemistry avidin-biotin binding, while not covalent, is essentially permanent. Also, since avidin has multiple binding sites for biotin, this can amplify the labeling. 

a labeled avidin binds to the biotin. This method has three incubation steps 

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Fig. 7.4 Direct avidin-biotin immunocytochemistry. An unlabeled 1° antibody binds to the antigen and a 2° antibody labeled with biotin binds to the 1° antibody.

Fig. 7.5 Indirect avidin-biotin immunocytochemistry. Following the 1° antibody binding to the antigen and a 2° antibody labeled with biotin binds to the 1° antibody. Unlabeled avidin is incubated in a third step and binds the biotin-labeled 2° antibody with several biotin-binding sites still unoccupied. In a fourth step, labeled biotin is bound to the avidin...

This method, either standard or elite (increased molar ratio), remains the forefront of much of the immunocytochemistry being performed today, and will be the main subject of this chapter. There are new methods, though, that are being used with increased frequency, such as the labeled-avidin binding method, sometimes called the streptavidin-binding method, and a newer catalyzed amplification method that uses avidin, biotin, peroxidase, and a biotinyl tyramide to achieve even more sensitivity. These methods will be discussed at the end of this chapter. 

Key Words Immunocytochemistry immunogold silver enhancement immunofluorescence immunoenzyme avidin biotin IGSS PAP APAAP epipolarization. 

Fig. 4. Immunocytochemistry of formalin fixed 147L) cell cultures using a-PR-A anti-receptor MAb (left) and a control MAb (right). Immunocytochemistry was performed with T47D breast cancer cells grown as monolayers in chamber slides. Cells were fixed for 15 min with 3.7% buffered formalin, followed by a permeabilization step with Triton X-100 (0.1%) for antibody penetration into the cell. Immunocytochemistry was performed by the indirect avidin-biotin immunoperoxidase method using diaminobenzi-dine as the chromagen.

Use of avidin-biotin molecules in immunocytochemistry dramatically increases the detection sensitivity and greatly increases the flexibility of labels. The power of avidin-biotin is their ability to bind with extremely high affinity to each other. In addition, the ligand (biotin) and receptor (avidin) are unique for each other. There are multiple ways that labels can be conjugated to avidin or biotin and then used in immunocytochemistry. This section will introduce these molecules and show a several methods for their use in immunocytochemistry. 

One advantage of indirect avidin-biotin method is an amplification of the detection system. For example, after biotin conjugated 2° antibody, avidin is incubated, binding to the available biotins. After the remaining free avidin is rinsed off, a biotin conjugated to 488 fluorophore is added and it binds to the remaining sites on the avidin. With this method, amplification occurs because any avidin can be attached to multiple biotins conjugated with either fluorescent or enzyme. This method requires two additional incubation steps of the indirect immunocytochemistry with fluorescent-labeled 2°. 

Avidin-biotin complex (ABC) uses a reagent made from avidin, biotin, and HRPfor a much larger increase in detection sensitivity (Hsu et al., 1981). ABC is based on the molecular complex that is made by mixing HRP-bound biotin with an excess of unlabeled avidin (Fig. 7.6). The proportions of biotin HRP and avidin are critical for developing the complex size that is needed for immunocytochemistry. The vendor. Vector Laboratories, produces an ABC reagent that is easy to use and consistent. 

Fig. 7.6 Avidin-biotin complex (ABC). To increase the number of HRP enzymes bound to a 1° antibody an avidin-biotin complex (ABC) is used. To make the complex, first multiple biotins are bound to HRP and then this is incubated with a dilute avidin. All of the HRP-biotin reagents bind to avidin, generating complexes with unbound avidin still available. This reagent is now used in ABC immunocytochemistry...

Fig. 7.7 Avidin-biotin complex (ABC) immunocytochemistry. After the 1° antibody binds the...

Low resolution occurs when enzymes (i.e., HRP), localized in the microscope generate a reaction product that spreads from the site of the enzyme and decreases the detection resolution. An example of lowest detection resolution would be ABC immunocytochemistry, because an enzyme is the label and it is bridged to the primary antibody by avidin-biotin (Fig. 9.1a, number 2, Low resolution). Any additional molecules that bind to the 1° antibody and bridge the label away from the 1° antibody will decrease the detection resolution. In planning experiments, resolution is important when antigens are localized to a discrete location. 

Avidin-biotin complex (ABC) - a reagent made from avidin, biotin, and HRP, which dramatically increases detection sensitivity for immunocytochemistry. 

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