Which statement about indirect enzyme immunoassay is false?

• A. The first antibody has the enzyme label.
• B. Color is directly proportional to the amount of patient antigen present.
• C. All reagents are added together.
• D. Enzyme specificity is essential.

Answer: (A)

In indirect ELISA the enzyme label sits on a secondary antibody, not on the first antibody. The plate is coated with antigen, the patient’s antibody (the first antibody) binds to that antigen, and then a secondary, enzyme-labeled antibody binds to the patient antibody. The color signal comes from the enzyme reacting with a substrate, and its intensity reflects how much patient antibody is bound.

So the statement that the first antibody has the enzyme label is not correct. The color readout is tied to the amount of patient antibody present, not the amount of antigen in the patient sample, and the assay is performed with sequential addition of reagents (not all at once). The enzyme’s ability to produce a color depends on a specific enzyme–substrate reaction, which is essential for generating a measurable signal.