In ELISA experiments, washing is a critical step that is often underestimated. Although it's not a chemical reaction, it plays a decisive role in the success of the entire procedure. Proper washing helps remove non-specifically adsorbed substances, which can interfere with the accuracy and reliability of the results.
Non-specific adsorption can occur during various stages of the experiment, especially when using serum samples or enzyme-labeled reagents. If these unwanted materials are not thoroughly removed, they can lead to increased background noise, false positives, or unreliable readings. For example, if the washing isn't done properly, the enzyme conjugate may remain on the plate, raising the blank value. In an indirect ELISA setup, un-washed IgG from the sample can also bind to the solid phase, causing interference with the labeled antibody.
To ensure effective washing, follow these steps:
1. After each incubation, carefully drain the liquid from the wells.
2. Add sufficient washing buffer to each well.
3. Let the buffer sit for about 2 minutes, gently shaking the plate if possible.
4. Drain the liquid again, then pat the plate dry on absorbent paper.
It's recommended to repeat this process 3–5 times, depending on the protocol and the type of ELISA being performed. The final wash is particularly important, as it ensures that all residual substances are removed before proceeding to the next step.
At Shanghai Hengyuan Bio, we emphasize the importance of meticulous technique in every ELISA experiment. If you have any questions or need further guidance, feel free to reach out to us. A well-executed washing step can make all the difference between a successful and a failed experiment.
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