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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 process can significantly affect the results. Common issues such as a "whiteboard" result, weak color development, or an uneven background often occur. Below, Shanghai Hengyuan provides detailed analysis of these problems. **Whiteboard Phenomenon** After completing all steps and adding the TMB substrate, the entire microplate shows no color. This could be due to several factors: - Reagents may have expired or were mixed from different batches. - Errors in adding detection antibody A or test antibody B, or the TMB solution. - Enzyme labels might have been contaminated or inactivated during washing or loading. - Diluents containing inhibitors like azide could interfere with the reaction. - Contaminated distilled water was used in the dilution process. - Washing solutions were not diluted properly, leading to over-concentration. - The enzyme label has low activity or potency. - Incorrect pH of the solution (should be between 7.2 and 7.4). **Low Color Sensitivity and Weak Signal** If the wells show only a very light color after TMB development, it may indicate: - Expired reagents or improper storage conditions. - Reagents, standards, or samples were not brought to room temperature before use. - Inadequate volume of reagent added or incorrect dilution ratio. - Enzyme standards may have been contaminated or inactivated. - Incubation time or temperature did not meet the required standards. - Excessive washing or improper washing concentration could remove bound antibodies. - TMB incubation time was too short. **Flower Plate Without Gradient Background** When the plate shows uniform color without a clear gradient, or a high background, it might be caused by: - TMB solution stored improperly and turned blue before use. - High incubation temperature or extended incubation time causing non-specific binding. - Incomplete washing as per instructions, especially if the plate wasn't washed enough. - Cross-contamination due to unclean pipette tips during sample loading. - Poor quality coating or detection antibodies, or insufficient sealing leading to unstable background. **Key Tips for Successful ELISA Experiments** To avoid common pitfalls, keep the following in mind: 1. Allow all reagents to reach room temperature before starting the experiment. 2. Ensure both coating and detection antibodies are highly active and target the same antigen. 3. Store protein-based reagents at -20°C and wash solutions at 2–8°C. 4. Mix each reagent thoroughly before use to maintain consistency. 5. Monitor and control reaction temperature and timing precisely. 6. Pay attention to washing procedures: number of washes, volume, duration, and strength. 7. Dilute concentrates as specified and maintain the pH between 7.2 and 7.4. 8. Avoid air bubbles when stopping the reaction. 9. Read the microplate within 2 minutes after stopping the reaction. By carefully following these guidelines, you can improve the accuracy and reliability of your ELISA results.

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