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. He mentioned that the results from the ELISA kit were not satisfactory and he was unsure what went wrong. Mr. Qian 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 antigen 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 in 10% calf serum. After incubating at 37°C for one hour, he washed the plate three more times, added the secondary antibody, and incubated for 40 minutes. Finally, he added TMB for color development. However, the results were unexpected—his negative control also showed color, and the difference between the positive and negative controls wasn't significant. He asked what could be the problem and whether it was due to improper washing. In response, the technical team from Shanghai Jinma analyzed the issue and provided several suggestions: 1. It's important to ensure that the washing process is thorough. If the plate is not properly washed, especially if there are "string holes" or incomplete washing, it can lead to contamination or background noise. We recommend washing the plate at least five times for both the primary and secondary antibody steps. 2. The blocking step appears to be a key issue. Since the negative control developed color, it suggests that the blocking solution might not have been effective enough. While OVA is commonly used, it may not always be the best choice depending on the system. You could consider alternative blocking agents like bovine serum albumin (BSA), casein, or even commercial blocking solutions available in the market. There are many resources online discussing the selection of appropriate blocking agents for ELISA. 3. Another point to consider is the incubation time. Although you've already incubated the plate at 37°C for two hours, sometimes extending this time slightly can improve signal-to-noise ratio. However, it’s also important to avoid over-incubation, which can cause non-specific binding. We hope these suggestions help you troubleshoot the issue. If you need further assistance or have more questions, feel free to reach out. Shanghai Jinma is committed to providing comprehensive support to our customers.

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