Analysis of the cause of the ELISA kit standard overflow

In the afternoon, Mr. Zhao from Liaoning University of Traditional Chinese Medicine reached out to Manager He at Shanghai Hengyuan to ask a question about an unusual "overflow" phenomenon observed in the ELISA standard product. Upon reviewing the issue, Manager He quickly forwarded it to the technical team. Within five minutes, the Shanghai Hengyuan technical department called Teacher Zhao to help investigate the possible causes behind the abnormal results. Here are some common reasons that could lead to such an overflow: 1. **Incorrect dilution of samples or standards**: If the sample or standard is not diluted properly, it can result in higher-than-expected readings. Always follow the instructions carefully when preparing your samples. 2. **Improper incubation time or temperature**: Incubation conditions must be strictly followed. Too much time or incorrect temperature can affect the reaction and cause inaccurate results. 3. **Failure to cover the microplate during incubation**: Covering the plate helps prevent contamination and evaporation. Each kit comes with enough plate covers, so make sure to use them as instructed. 4. **Incorrect detection wavelength**: Ensure that the spectrophotometer is set to the correct wavelength specified in the kit’s manual. A wrong setting can lead to faulty measurements. 5. **TMB substrate color development time too long**: Monitor the color development closely. The time may vary slightly depending on environmental factors like temperature. Following the recommended time is crucial. 6. **Inappropriate sample type**: Not all samples are suitable for every ELISA kit. Check the instruction manual for the acceptable specimen types. If your sample is outside the range, further optimization might be needed. 7. **Contamination of samples or standards**: Even minor contamination can lead to false positives or high readings. Handle all reagents and samples with care. 8. **Improper dilution of the conjugate**: Dilute the conjugate exactly as described in the manual. Over-concentration can cause excessive signal and result in an "overflow." 9. **Using the wrong diluent**: Only use the diluent provided in the kit. Using other solutions may interfere with the reaction and lead to unreliable results. Once the cause is identified, resolving the issue becomes much easier. The same applies to you—pay close attention to each step, follow the guidelines, and always read the instructions thoroughly before starting your experiment. At Shanghai Hengyuan, we hope your experiments go smoothly and yield accurate results!

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