Possible reasons are listed below:
- The reagents have expired
- Adding detection reagent A, B or TMB substrate was skipped
- Distilled water used for dilution was contaminated
- Enzyme inhibitor (such as Sodium azide) is present
- Wash buffer is concentrated which is not diluted in proportion.
- The activity and titer labeled enzyme is low.
- PH value of solution is incorrect. Normally it should maintained at 7.2-7.4.