1. Prepare all reagents, samples and standards;
2. Add 50µL standard or sample and 50µL Detection Reagent A to each well. Incubate 1 hours at 37°C;
3. Aspirate and wash 3 times;
4. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
5. Add 50µL Stop Solution. Read at 450nm immediately.