What is the basic principle of the ABC Method?

• A. Direct enzyme conjugation of primary Abs
• B. Biotin-streptavidin- peroxidase amplification; Secondary Abs conjugated with Biotin; Avidin-Biotin link; 2 Biotin molecules required
• C. Fluorescence-labeled detection only
• D. No amplification

Answer: (B)

The main idea behind the ABC method is signal amplification using the strong biotin–avidin (or streptavidin) interaction to bring enzyme activity to the antigen site. After the primary antibody binds its target, a biotinylated secondary antibody attaches to the primary. Then an avidin–biotin–peroxidase complex is applied, which binds to the biotin on the secondary. Because avidin (or streptavidin) can bind multiple biotin units and the secondary antibody can present several biotin groups, multiple peroxidase enzymes accumulate at the same location. When a chromogen is added, the enzyme catalyzes a color reaction, producing a much stronger signal than direct enzyme conjugation would. This amplification makes the method particularly sensitive and is why this approach is favored over non-amplified or non-enzymatic detection.