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, asking a question about an unusual "overflow" phenomenon observed with the ELISA standard product. Upon reviewing the issue, Manager He quickly escalated it to the technical team. Within five minutes, the Shanghai Hengyuan technical department called back to help analyze the possible causes behind the abnormal results. They listed several potential reasons that could lead to such an overflow in the ELISA test: 1. **Incorrect dilution of samples or standards** – If the sample or standard is not diluted properly, it can cause high OD readings and result in an overflow. 2. **Improper incubation time or temperature** – Incubation conditions must be strictly followed. Any deviation can affect the reaction and lead to 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 for this purpose. 4. **Incorrect detection wavelength** – Make sure the spectrophotometer is set to the correct wavelength as specified in the kit instructions. 5. **TMB substrate color development time too long** – The color development should be monitored closely. The recommended time may vary slightly depending on environmental factors like temperature. 6. **Inappropriate sample type** – Not all sample types are suitable for every ELISA kit. Always check the instruction manual for the applicable specimen range. 7. **Contamination of samples or standards** – Contaminated samples can lead to false positive results and cause an overflow. 8. **Improper dilution of the conjugate reagent** – Follow the instructions precisely when diluting the conjugate. An overly concentrated solution can lead to high OD readings. 9. **Using the wrong diluent** – Only use the diluent provided in the kit to prepare samples and standards. Using other solutions can interfere with the reaction. Once the cause was identified, resolving the issue became much simpler. This experience highlights the importance of following the protocol carefully and paying attention to details. It's crucial to read the ELISA kit instructions thoroughly before starting any experiment. At Shanghai Hengyuan, we hope all researchers have smooth and successful experiments. Let us know if you need further assistance!

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