The development of the enzymatic reaction is the same for conventional and sandwich ELISA. This method is appropriate for horseradish peroxidase as a reporter enzyme. A different substrate is required if alkaline phosphatase is used as the reporter enzyme (Harlow and Lane, 1988), together with a different wavelength for determining the absorbance, but the overall procedure is the same.

1. Prepare TMB substrate solution:
1.1.  100 ml   water,
1.2.  1 ml                10mg/ml TMB (33’55’TetraMethylBenzidine) in DMSO (DiMethylSulfOxide),
1.3.  10 ml              1M Na Acetate (pH 5.8 with 1.0M citric acid),
1.4.  20 µl               30% H2O2.
2. Add 200 µl substrate solution to each well.
3. Let colour develop for 10-15 minutes .
4. Add 50 µl 3M H2SO4 to terminate the reaction.
5. Immediately read the absorbance at 450nm (the termination reaction will continue to develop colour).