ELISA in its indirect format is used to detect antibodies in patient serum. What is the role of the secondary antibody conjugated to enzyme in indirect ELISA?

• A The secondary antibody is a species-specific anti-human immunoglobulin conjugated to enzyme (HRP or AP); it binds to all classes of patient IgG/IgM that have already bound the antigen, amplifying signal by allowing multiple secondary antibody molecules per primary antibody ✓
• B The secondary antibody is the specific antigen-coated antibody that captures the target antigen from patient serum onto the plate
• C The secondary antibody is a monoclonal anti-HRP antibody that stabilizes the enzyme-substrate reaction
• D The secondary antibody is the blocking reagent (BSA or milk protein) that reduces non-specific binding

Explanation

In indirect ELISA: antigen is adsorbed to the plate; patient serum (primary antibody) is added and binds antigen if antibodies are present; after washing, a secondary antibody — typically a polyclonal anti-human IgG (or IgG+IgM) conjugated to an enzyme (horseradish peroxidase or alkaline phosphatase) — is added; this secondary antibody binds the Fc region of all primary antibody molecules that have bound antigen. Because multiple secondary antibody molecules can bind each primary antibody and each enzyme molecule amplifies the signal, indirect ELISA has high sensitivity. The secondary antibody is NOT the capture antibody, NOT anti-HRP, and NOT a blocking agent.

Reference: Ananthanarayan & Paniker's Textbook of Microbiology, 11th ed.

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