No Signal (After adding the TMB substrate, all wells are colourless)

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.
Weak Signal (After adding TMB substrate, all wells are weakly coloured)

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.
Lack of a Colour Gradient (All wells of the plate have the same strong or weak colour signal)
  • 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.
Things to pay attention to before beginning the experiment
  • 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.

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