In the ABC method, what is the role of the secondary antibody?

• A. It binds to the primary antibody
• B. It binds directly to the antigen
• C. It binds to avidin
• D. It binds to the substrate

Answer: (A)

In the ABC method, the key role of the secondary antibody is to recognize and bind to the primary antibody that is attached to the antigen. This creates a binding site for the avidin-biotin–HRP complex, which provides the signal for visualization. The secondary antibody is chosen to target the species and class of the primary antibody, binding the constant region rather than the antigen itself. This is why it doesn’t bind directly to the antigen or to the substrate; the signal comes from the enzyme in the avidin–biotin complex that is recruited after the secondary antibody has bound the primary.