Wash buffer was contaminated? Cross contamination with other reagents? Well plate insufficiently shake after wash
Dilution buffer was contaminated? Clean and colorless on reagents when it is
in preparation? 3.
Contaminated on incubation Use new plate sealer when it is on
incubated? Any liquid or dirty on well plate? 4. TMB
Substrate Solution was contaminated Any change on the color of TMB? Substrate incubation carried out in light? 5.
Pipette was contaminated? Cross-used when it is in test? 6.
Reagents preparation Correct and proper dilution rate for all
Keep the well plate completely dry at any time?