The use of avidin-biotin interaction in immunoenzymatic techniques provides a simple and sensitive method to localize antigens in formalin-fixed tissues. Among the 5everal staining procedures available, the ABC method, which involves an application of biotin-labeled secondary antibody followed by the addition of avidin-biotin-peroxidase complex, gives a superior result when compared to the unlabeled antibody method. The availability of biotinbinding sites in the complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxi-Literature Cited

A highly sensitive immunoenzymatic technic is presented. The method involves three sequential steps: (1) primary antibody, (2) biotin-labeled secondary antibody, and (3) avidin-biotin-peroxidase complex. Avidin, an egg white protein, has four binding sites for the low-molecular-weight vitamin biotin. Many moieties of biotin can be coupled to the peroxidase molecule. Thus, since a relatively large amount of avidin is incubated with biotin-labeled peroxidase, avidin serves as a link between biotin-peroxidase molecules; in turn, biotin-peroxidase serves as a link between avidin molecules. Consequently, this large lattice-like complex with biotin-binding capability can be attracted to the sites of biotin-labeled antibody, producing a superior staining sensitivity. Several commercially available radioimmunoassay antibodies (e.g., antiglucagon, prolactin, gastrin, growth hormone, and thyroid-stimulating hormone antibodies) were tested for immunohistochemical staining. The unlabeled antibody peroxidase-antiperoxidase method fails to stain gastrin or thyroid-stimulating secretory cells when using these antibodies, and a relatively high antibody concentration is required to produce a positive reaction for glucagon, prolactin, and growth hormone. In contrast, the avidin-biotin-peroxidase complex method successfully demonstrates polypeptide hormones even when antibodies are diluted 20 to 40 times.

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Avidin has an extraordinary affinity for the small-molecule vitamin biotin. Covalently coupling biotin or avidin to peroxidase molecules does not interfere with their normal biochemical functions. The avidin or biotin molecules, either peroxidase conjugated or unconjugated, can be brought to the antigen sites by means of an antiavidin antibody. Several immunohistochemical staining technics based on this principal have been described. The method utilizing an avidin-biotin-peroxidase complex was found to be more sensitive than the unlabeled antibody (PAP) method. This method involved four sequential staining procedures: (1) primary antibody (goat anti-human antigen); (2) secondary antibody (rabbit antigoat IgG) added in relative excess; (3) goat antiavidin antibody; (4) avidin-biotin-peroxidase complex. The applications of this technic are discussed.