Immunoassay method for thyroxine using a homogeneous assay where fluorogenic substrate-labeled thyroxine competes with patient T4 for antibody; only unbound labeled T4 forms a fluorescent product.

• A. Chemiluminescence
• B. Fluorescent polarization immunoassay (FPIA)
• C. Microparticle enzyme immunoassay (MEIA)
• D. Fluorescent substrate-labeled inhibition immunoassay

Answer: (D)

The main idea is a homogeneous competitive (inhibition) immunoassay using a fluorogenic tracer. In this format, a labeled thyroxine tracer competes with the patient’s T4 for a limited antibody. Because only the tracer that remains unbound to the antibody can be converted by the enzyme into a fluorescent product, the fluorescence signal tracks how much tracer stays free. As the patient’s T4 increases, more tracer is sequestered by the antibody, leaving less unbound tracer to generate fluorescence, so the signal changes in a way that can quantify T4. This fits the description of a fluorescent substrate-labeled inhibition immunoassay, a homogeneous method with competitive binding and a fluorescence readout. The other methods involve different readouts or formats (such as fluorescence polarization, solid-phase enzyme formats, or chemiluminescent detection) that don’t match this specific mechanism of signal generation.