You have weak fluorescence in both direct and indirect FA runs for the same patient. To determine whether low signal is due to low antigen abundance vs low antibody affinity in serum, which follow-up experiment would best help you discriminate between the two possibilities?

• A. Switch to a different fluorescent dye only on the secondary antibody and assume that a color change will inherently fix issues related to antigen abundance and primary antibody affinity.
• B. Conclude immediately that the patient is immunodeficient and report results as undetectable for both antigen and antibody without any further validation experiments.
• C. Spike the patient sample slide with a known quantity of the target antigen and rerun the direct FA to test antibody functionality; concurrently test patient serum against a high-concentration lab antigen to check for primary antibody binding in the indirect FA.
• D. Ignore the weak signals and rerun both assays at a later date with no controls, because repeating without changes will average out the variability and reveal the true result.

Answer: (C)