The ELISA (Enzyme-Linked Immunosorbent Assay) kit needs to be kept in the experiment because it's a solid-phase immunoassay, where antigen-antibody interactions occur only on the surface of a solid support. For example, in a sandwich ELISA, where antibodies are coated onto a microplate, the sample added to each well doesn’t have equal access to bind with the immobilized antibody. Only the antigens closest to the well walls can directly interact with the antibodies. This process is gradual and reaches equilibrium over time, which is why incubation is necessary for the reaction to complete effectively.
Typically, there are two main antigen-antibody reactions in an ELISA: one after adding the sample and another after adding the enzyme-conjugated secondary antibody. Incubation is crucial for these steps to ensure proper binding and signal development.
To maintain optimal conditions during incubation, most labs use either a water bath or a wet incubator. If using a water bath, the ELISA plate should be placed inside a box with the bottom of the plate just touching the water surface to help achieve even temperature distribution. To prevent evaporation, the plate should be covered with plastic wrap or sealing film. In some cases, the plate can float on the water surface for better heat transfer.
If an incubator is used, the ELISA plate should be placed in a metal wet box that contains a layer of damp gauze at the bottom. The box should be pre-warmed to the desired temperature before placing the plate inside. It’s important not to stack plates, as this could affect temperature uniformity. At lower temperatures, pre-warming the box becomes even more critical.
For room temperature incubation, the ELISA plate should be placed flat on a stable surface, such as a lab bench. The temperature and time must be strictly controlled according to the manufacturer's instructions. Room temperature is typically defined as 20–25°C, but specific requirements may vary depending on the kit. When working at room temperature, it's best to handle no more than two plates at a time to avoid inaccuracies in timing and temperature control.
Proper incubation ensures accurate and reproducible results, making it a vital step in the ELISA procedure.
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