ELISA experiment must learn: washing method - Database & Sql Blog Articles

After the plate is washed, it removes non-specific binding—substances that do not react with your target on the board. This step is crucial because if you leave residues in the liquid, they can interfere with your results. You need to wash the plate multiple times to ensure that all unbound components are removed. However, the substances that specifically bind to your target, such as primary antibodies or secondary antibodies, will stay firmly attached and won’t be washed away easily. If you skip this step, these residuals could lead to false positives and increase background noise, which is a common issue in ELISA experiments. The washing process helps reduce background signals from unbound antibodies, thereby improving the signal-to-noise ratio of your analysis.

There are two main methods for washing the plate:

1. Automatic Washing: If you have access to an automated washer, it's recommended to use it after becoming familiar with its operation. It ensures consistency and efficiency in the washing process.
2. Manual Washing: When using manual techniques, avoid touching the plate directly. Place several layers of absorbent paper on the bench, then carefully aspirate or remove the liquid from the microplate. It’s important to be gentle to prevent any damage. Use at least 0.3 ml of wash buffer per well, let it soak for 1–2 minutes, and repeat the process as needed. This method requires more attention but is effective when automation isn't available.

ELISA (Enzyme-Linked Immunosorbent Assay) is a powerful technique that combines the high specificity of antigen-antibody interactions with the sensitivity of enzyme-catalyzed reactions. Here are some key advantages of using ELISA:

• The enzyme-labeled reagents allow for highly sensitive detection, making it possible to identify even small amounts of target molecules.
• It enables both the localization and quantification of antigens or antibodies at the cellular or subcellular level, down to microgram or even nanogram levels.
• Due to the strong specificity of the antigen-antibody interaction, ELISA minimizes cross-reactivity and provides reliable results.