Alternative Block ELISA Blocking Buffer

1. Allow product to equilibrate to room temperature (25°C) prior to use., 2. Add 100 µL to each well. When reacting with peroxidase, a soluble blue reaction product develops that can be read at 370 nm or in a range of 620-650 nm. The reaction product is stable for 1 hour., 3. The reaction can be stopped using an appropriate stop solution, such as Stop Solution for TMB Substrates (STOPT), for endpoint assays. STOPT changes the chromogen from blue to yellow and the absorbance to 450 nm. For best results, sample absorbance should be monitored and read before values exceed 2.0 OD units. STOPT can increase the absorbance up to 3-fold, so the reaction should be stopped when absorbance values reach approximately 0.7 OD units.