Shanghai Hengyuan analyzes common problems in ELISA experiments for you - Database & Sql Blog Articles
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EL-C1600N100013-B
Heng Jiaxing AO3401
Probe domestic double-head probe 038-BB-5.7L The shape of the two ends of the needle is outside the pointed needle
ELISA experiments are widely used in biological research due to their high sensitivity and specificity. However, for beginners, even small mistakes during the procedure can significantly affect the final results. Common issues include a "whiteboard" effect, weak color development, or uneven background. In this article, we will explore these problems and provide practical solutions to help improve your ELISA performance.
The Whiteboard Phenomenon
This occurs when, after adding the TMB substrate solution, all wells on the microplate remain completely colorless, indicating no signal was detected.
Possible Causes:
Reagents have expired or were stored incorrectly.
Incorrect dilution or mixing of reagents between different batches.
Mistakes in adding detection antibody (A) or test antibody (B), or TMB substrate.
Enzyme labels may be contaminated or inactivated during washing or sample loading.
Diluent containing inhibitors such as azide or contaminated distilled water.
Washing buffer not diluted properly or washed too many times.
Low enzyme activity or incorrect pH of the solution (should be 7.2–7.4).
Low Color Sensitivity and Weak Signal
In this case, the color developed is very light, leading to low sensitivity and poor detection capability.
Possible Causes:
Expired reagents or improper storage conditions.
Reagents, standards, or samples not brought to room temperature before use.
Incorrect volume or dilution of reagents added.
Contamination or inactivation of enzyme-labeled antibodies during washing or loading.
Incorrect incubation time or temperature.
Improper washing steps, excessive washes, or over-washing affecting signal.
Insufficient TMB development time.
Flower Plate with No Gradient Background
This issue arises when the wells show color but lack a clear gradient, and the background is unusually high.
Possible Causes:
TMB substrate was improperly stored and turned blue before use.
Over-incubation at high temperatures causing non-specific binding.
Failure to follow washing instructions, especially during plate washing.
Contamination from pipette tips during sample addition.
Low activity or concentration of coating or detection antibodies.
Insufficient sealing, incomplete washing, or improper coating of the plate.
Key Tips for Successful ELISA Experiments
To avoid these common pitfalls, consider the following best practices:
Allow all reagents to reach room temperature before starting the experiment.
Ensure that both the coating and detection antibodies are highly active and target the same antigen.
Store protein-based reagents at -20°C and washing solutions at 2–8°C.
Thoroughly mix each reagent before use to ensure consistency and accuracy.
Control reaction temperature and timing precisely.
Pay close attention to washing steps—number of washes, volume of wash solution, and washing duration.
Dilute concentrated reagents according to instructions and maintain a pH between 7.2 and 7.4.
Avoid air bubbles when stopping the reaction.
Read the plate within 2 minutes after stopping the reaction to ensure accurate results.
By carefully following these guidelines, you can significantly improve the reliability and reproducibility of your ELISA experiments.