Avidin-Biotin Complex ABC Immunocytochemistry

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. 

The ABC contains numerous HRP molecules for label generation, and unbound avidin available to bind a biotin 2° antibody. 

ABC is a very popular way of amplifying the HRP label for light microscopy. For example, an experiment uses a rabbit anti-antigen H antibody and a 2° goat antirabbit antibody labeled with biotin (Fig. 7.7). When the ABC complex is added, free avidin on the surface of the complex binds to biotin on the antibody, which carries many more HRP molecules than a single HRP-labeled 2° antibody. Vector Laboratories makes ABC kits that use dropper bottles to measure reagents. Such kits are almost foolproof. 

While the use of the ABC increases detection sensitivity by adding HRP enzymes (Fig. 7.7) to the labeling process, it does not change either the number of the 1° 

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

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