Which workflow correctly describes the five main steps of an indirect ELISA and the rationale for how the assay demonstrates prior exposure to an antigen?

• A. Coat wells with known antigen → block unoccupied surface → add patient serum so any specific primary antibodies bind the antigen → add enzyme-linked secondary antibody that recognizes human antibodies → add substrate; a color change indicates the patient has circulating antibodies from prior exposure or vaccination.
• B. Coat wells with whole killed pathogen → skip blocking to maximize binding → add patient serum → perform multiple alternating washes of enzyme and substrate → color development indicates acute infection only and cannot reflect prior exposure.
• C. Coat wells with random serum proteins → add enzyme-labeled antigens from pathogen → block wells to stop reaction → add patient serum to compete off labeled antigens → read color; this competition indicates exposure only if labeled antigen completely displaces serum antibodies.
• D. Coat wells with capture antibodies to bind patient antibodies directly → block → add enzyme-linked primary antigen → add substrate; a color change indicates presence of antigen rather than prior exposure to antigen.

Answer: (A)