The results of the ELISA kit will be analyzed like this - Database & Sql Blog Articles

In the past few days, our sales representative had an online conversation with Mr. Qian, a teacher from Shanghai Jiao Tong University. Mr. Qian mentioned that the results from the ELISA kit were not as expected, and he was unsure of what went wrong. He explained that he synthesized an 8-amino-acid antigen short peptide and immunized an animal after coupling it with BSA. One month later, he tested the antibody titer against the short peptide using ELISA. The procedure he followed was as follows: he dissolved the antigen in carbonate buffer, coated the plate, and left it at 4°C for 18 hours. Then, he blocked the plate with 1% OVA at 37°C for two hours, washed it three times, and diluted the serum to be tested with 10% calf serum. After incubating at 37°C for one hour, he washed the plate three more times, added the secondary antibody, and incubated it for 40 minutes at 37°C. Finally, he added TMB reagent for color development. However, the results were inconsistent—his negative control also showed color, and the difference between the positive and negative was not significant. He asked, “What could be the problem? Is it due to washing?” Shanghai Jinma’s technical team responded by analyzing the possible causes: 1. **Washing Procedure**: It is crucial to ensure that the washing step is thorough. If the plate is not properly washed, residual reagents or unbound antibodies may lead to background noise. It's recommended to wash the plate at least five times after each primary and secondary antibody incubation. 2. **Blocking Solution**: The fact that the negative control developed color suggests a potential issue with the blocking step. While OVA is commonly used, it might not be the best choice in all cases. Other blocking agents like BSA or casein could provide better results. It’s worth checking which blocking solution works best for your specific assay. 3. **Incubation Time and Temperature**: Although you mentioned incubating at 37°C, extending the incubation time for the primary and secondary antibodies might improve signal detection. Some protocols recommend longer incubation periods for better binding. We hope this helps clarify the issues you’re facing. If you have any further questions or need additional support, feel free to reach out. Shanghai Jinma is here to assist you every step of the way.

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