In a noncompetitive enzyme immunoassay, if a negative control shows color, which is a possible explanation?

• A. No reagent was added.
• B. Washing steps were incomplete.
• C. The enzyme was inactivated.
• D. No substrate was present.

Answer: (B)

In a noncompetitive enzyme immunoassay, color development comes from enzyme activity that only occurs when the detection antibody bound to antigen is properly washed away if no antigen is present. If a negative control shows color, it points to a signal coming from reagents that should have been removed. The most plausible cause is incomplete washing, which leaves unbound or loosely bound enzyme-labeled detection antibodies in the well. Those residual enzymes can still react with the substrate and produce color even without the target antigen, giving a false-positive signal in the negative control.

If no reagent were added, or the enzyme were inactivated, or no substrate were present, you wouldn’t see color in the negative control—the other options would prevent color formation.