The ELISA kit must be kept in the experiment because it is a solid-phase immunoassay, where antigen-antibody binding occurs only on the surface of a solid support. For example, in a sandwich ELISA, the antibody is first coated onto the plate. When the sample is added, not all antigens in the solution have an equal chance to bind to the immobilized antibody. Only the antigens that are closest to the well walls can directly interact with the antibody. This process gradually reaches equilibrium, so incubation is necessary for the reaction to complete effectively.
In typical ELISA procedures, there are two antigen-antibody reactions: one after adding the sample and another after introducing the enzyme-labeled secondary antibody. To maintain consistent conditions during these steps, proper incubation methods are essential. While some specialized ELISA instruments use electric heating blocks, most experiments rely on water baths. The ELISA plate should be placed in a water bath box with its bottom in direct contact with the water to ensure rapid temperature equilibration. To prevent evaporation, the plate should be covered with plastic wrap or sealing film.
If an incubator is used instead, the ELISA plate should be placed inside a wet box. The box should be made of a material with good heat transfer, such as metal, and a damp cloth should be placed at the bottom. The plate is then positioned on top of the wet cloth. The wet box should be pre-warmed in the incubator to the desired temperature, especially when working at lower temperatures. Whether using a water bath or a wet box, the plates should not be stacked to ensure uniform temperature distribution across each well.
For room temperature incubation, the ELISA plate should be placed flat on a stable surface. It's crucial to strictly control both temperature and time, as deviations can affect results. To ensure accuracy, it’s recommended that one person does not handle more than two plates at a time. Maintaining precise conditions throughout the incubation process is vital for reliable and reproducible results.
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