Antigens localized using avidin-biotin

The procedure given here summarizes the localization of tissue antigens using a primary antibody, biotinylated secondary antibody, avidin-biotin complex, and DAB chromagen on fresh frozen brain... 

If cellular localization of the antigen-antibody complex is not required, enzyme immunolabeling can be performed on cells adherent to a microtiter plate, and the color change resulting from the enzymatic reaction can be detected as a change in absorbance with an automatic plate reader (see Chapter 28). Biotinylation of antibodies and the use of the avidin-biotin complex has further extended the versatility and sensitivity of the enzymatic techniques (see Chapters 7 and 25-27). Most recently, the principles behind these techniques have been applied in combination with in situ hybridization techniques. Using nucleic acid-antibody complexes as probes, specific DNA or RNA sequences can be localized (see Chapters 46 9). 

As part of the localization procedure, the antigen-bound primary antibody is detected by a system that permits direct obseiwation of the site of antibody binding. A number of detection systems are available to enhance sensitivity and to visualize primary antibody bound in situ to target antigen. Most of them use either a radiolabeled second antibody and visuahzation by autoradiography, or an enzyme, fluorescent, or electron dense probe coupled to a linking second antibody, protein A, or avidin-biotin amplification system (l.,2). 

The simultaneous localization of antigens may be complicated if monoclonal primary antibodies are used for both epitopes. Monoclonal antibodies are often of the IgGl subclass, which cannot be distinguished by secondary antibodies. Hapten-labeling or biotin-avidin bridging could be of considerable value to circumvent this problem. 

FIGURE 1.8 (A) Direct biotin-avidin method. The primary antibody is linked to biotin (B) an avidin-peroxidase-conjugate (A-Px) is then added. (B) Indirect biotin-avidin method. Used for monoclonal antibodies, the primary antibody is not conjugated its localization is detected by a biotinylated secondary antibody. Boxed asterisk represents antigen determinant on primary antibody. Px, peroxidase label A, avidin B, biotin. From Taylor CR, Cote RJ, eds. Immunomicroscopy A Diagnostic Tool for the Surgical Pathologist. 3rd ed. Philadelphia Elsevier 2005 21. 

Originally described several years ago for use in immunoassay systems (36), biotinylated tyramine amplification has recently been adapted for immuno-cytochemical use (37). The system is based on the ABC method. H2O2 is catalysed to H2O and oxygen-free radicals by HRP as usual. This then reacts with biotinylated tyramine to produce activated biotinylated tyramine. which binds to nearby proteins in a highly localized manner. The high concentrations of biotin thus produced can then be detected by the application of one additional layer of avidin-reporter conjugate. The system has been used to demonstrate antigens previously only detectable in frozen sections, and to increase primaiy antibody dilution. Several manufacturers produce kits that are based on the system (CSA from Dako Ltd. and TSA from NEN life Science Products), or home-made biotinylated tyramine can be produced. 

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