In immunoperoxidase staining using the 3-step indirect method, which antibody is specific for the antigen?

• A. Primary monoclonal antibody
• B. Secondary antibody
• C. Tertiary antibody
• D. Blocking serum

Answer: (A)

The key idea is that the antigen in the tissue is recognized directly by the primary antibody. In a 3-step indirect immunoperoxidase staining, the primary antibody is chosen for its ability to bind specifically to the target antigen—often a monoclonal antibody because it recognizes a single epitope, providing precise specificity. The subsequent steps involve antibodies that bind to the primary antibody to detect and amplify the signal: the secondary antibody recognizes the primary antibody’s species, and a tertiary antibody can further amplify by binding to the secondary. Blocking serum’s job is to prevent non-specific sticking, not to bind the antigen. So the antibody that is specific for the antigen is the primary antibody.