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• 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 the uniformity and accuracy of each reagent, mix all components 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.
• Washing technique should be kept consistent throughout the experiment and 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.

Possible reasons:

• Degradation of the standard due to time or improper storage
• Standard curve dilutions were prepared incorrectly
• Pipetting errors leading to inconsistent concentrations
• Standard curve data was plotted improperly or without using appropriate software

Possible reasons:

• The reagents have expired - check packaging for expiration date
• Adding detection reagent A, B or TMB substrate was skipped
• The distilled water used for dilution was contaminated
• An enzyme inhibitor (such as Sodium azide) is present
• Wash buffer is too concentrated and has not been appropriately diluted
• The labelled enzyme is contaminated or inactive
• pH value of solution is too high or low. All solutions should be maintained at pH 7.2-7.4 for the entire experiment

Possible reasons:

• The reagents have expired or have not been stored in the correct conditions - check packaging for expiry date
• 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 was not incubated long 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

Possible reasons:

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

Possible Reasons:

• Wells were washed inconsistently or insufficiently at one or more steps 
• Reagents were mixed or prepared inconsistently 
• One or more solutions were contaminated
• Bubbles formed in wells