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Far Mu biological detailed ELISA test kit immunoassay sandwich method - Database & Sql Blog Articles
The ELISA test kit is a widely used immunoassay technique based on the sandwich method. It involves detecting specific antigens or antibodies by binding them to a solid phase, typically a microplate. Proper incubation conditions are essential for accurate and reliable results.
When using a water bath, the ELISA plate should be placed in such a way that its bottom touches the water surface to ensure even temperature distribution. To prevent evaporation, it's recommended to cover the plate with plastic wrap or sealing film. The plate can then float on the water surface. Alternatively, if an incubator is used, the plate should be placed inside a wet box made of a material with good thermal conductivity, such as metal. A layer of damp gauze should be placed at the bottom of the box, followed by the ELISA plate. The wet box must be pre-warmed to the desired temperature, especially when working at lower temperatures.
Whether using a water bath or a wet box, it’s important not to stack the plates to ensure uniform temperature across all wells. For room temperature incubation, the ambient temperature should be strictly maintained within the range specified by the manufacturer—typically 20–25°C. The ELISA plate should be placed flat on a stable surface during this process. Accuracy in both time and temperature is crucial, so it's advisable that one person handles no more than two plates at a time to avoid errors.
Since ELISA is a solid-phase immunoassay, the interaction between antigen and antibody occurs only on the surface of the microplate. This means that not all antigens in the solution will have an equal chance of binding to the immobilized antibodies. Only those closest to the well walls can directly interact with the antibodies. This process requires diffusion and equilibrium, which is why incubation is necessary for the reaction to complete properly. The same principle applies to the subsequent binding of enzyme-labeled antibodies to the solid-phase antigens.
Common incubation temperatures include 43°C, 37°C, room temperature (20–25°C), and 4°C (refrigerator temperature). Among these, 37°C is the most commonly used in laboratories because it supports optimal antigen-antibody binding. Studies have shown that most reactions reach their peak after 1–2 hours at this temperature. Raising the temperature can speed up the reaction, but higher temperatures may lead to non-specific binding or denaturation. Some tests use 43°C, but this is not always ideal. At 4°C, the reaction is more thorough, and overnight incubation in the refrigerator allows maximum precipitation. However, due to the extended time required, this method is rarely used in routine ELISA procedures.
In summary, proper handling, accurate temperature control, and sufficient incubation time are critical for successful ELISA testing. Following these guidelines ensures reliable and reproducible results, making the ELISA test kit a powerful tool in immunological research and diagnostics.