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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.

Possible reasons are listed below:

• The reagents have expired or have not been stored in the correct conditions.
• The reagents, standard, and/or sample were not warmed to room temperature prior to conducting the experiment.
• Incorrect amounts and/or dilutions of reagents were used.
• One or more solutions used were contaminated.
• The labelled enzyme is contaminated or inactive.
• TMB substrate chromogenic time is not enough.
• Washing procedure problems, i.e.:
  • Wash buffer too concentrated.
  • Wells were washed too many times.
  • Wells were washed too vigorously.
  • Wells were washed too long each time.

• TMB substrate was not stored in a cool dark place and protected from light exposure.
• The incubation time was too long (which can lead to strong, nonspecific adsorption).
• Wells were not washed adequately during the experiment.
• Cross contamination from pipette tips or contaminated reagents.
• Low concentration or low activation of the coated or detection antibody.

• All reagents in the kit should be stored according to the manual instructions.
• Before beginning the experiment, all components should be brought to room temperature.
• To ensure uniformity and accuracy of each reagent, mix all components of the kit before use.
• Keep the temperature stable at room temperature throughout the entire experiment.
• The pH should be maintained between 7.2-7.4 for the entire duration of the experiment.
• Ensure all concentrated reagents are appropriately diluted prior to beginning the experiment.
• The washing technique should be kept consistent throughout the experiment and should be done according to the instructions in the manual.
• Avoid creating bubbles in solutions to ensure uniformity of all reactions.
• Take the reading of the microtiter plate 2 minutes after adding the stop solution.