In the ABC detection system, what forms the large complexes that generate signal?

• A. Avidin and biotinylated enzyme
• B. Primary antibody and antigen
• C. Secondary antibody and antigen
• D. Avidin and primary antibody

Answer: (A)

Signal amplification in the ABC detection system comes from forming large complexes of avidin bound to biotinylated enzyme. Avidin has four high-affinity biotin-binding sites, so when a biotinylated enzyme (such as HRP) is present, avidin can assemble a bulky complex that localizes at the site where the antibody has bound the antigen. This ABC complex brings multiple enzyme molecules to the site, producing a strong signal when substrate is added. The other interactions (antigen with antibody, or antibody with antigen) are part of binding, but without the avidin-biotin-enzyme assembly, there isn’t the amplified enzymatic signal.