One of the most important factors that can determine the success or failure of an ELISA experiment is the incubation process. As a solid-phase immunoassay, ELISA relies on the antigen-antibody binding reaction taking place on a solid surface. For this reaction to be complete, the antigens or antibodies in the liquid phase must bind effectively to their respective partners on the solid phase. This requires specific temperature and time conditions to ensure accuracy.
Shanghai Jinma Biotechnology Co., Ltd. emphasizes that proper attention should be given to the incubation step when using ELISA kits. The incubation time is inversely related to temperature — the higher the temperature, the shorter the required time. Commonly used incubation temperatures include 37°C and room temperature (20–25°C), with some protocols also using 43°C or 2–8°C. However, changing these parameters arbitrarily without following the manufacturer's guidelines can lead to unreliable results.
It’s essential to follow the recommended incubation times and temperatures specified by the kit instructions. Some operators may adjust the protocol based on personal preference, which can introduce errors into the experiment. Different ELISA kits are designed for specific conditions, so deviating from them may result in inconsistent or inaccurate outcomes.
The incubation method typically involves using a dedicated heating block or water bath provided with the ELISA instrument. For optimal performance, it's recommended to use an incubator. Place the ELISA plate inside a humidified box with a wet gauze at the bottom to maintain moisture. Avoid stacking plates to ensure even temperature distribution across all wells. When working at room temperature, make sure the ambient temperature remains within the specified range (usually 20–25°C) as outlined in the instructions.
It's also important to measure only one or two plates at a time to maintain consistency and avoid human error. Shanghai Jinma Bioassay Kits are made from high-quality imported materials and come with a specially formulated diluent. These kits have undergone rigorous cross-reactivity, interference, and stability testing, ensuring excellent performance with low intra- and inter-assay coefficients of variation (less than 10%).
ELISA is widely used for both qualitative and quantitative analysis of various immunological markers, including cytokines, chemokines, growth factors, post-phosphorylation targets, and immunoglobulins. With its high sensitivity and cost-effective design, the ELISA method continues to be a reliable choice in research and diagnostic settings.
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