Can make your ELISA kit better than expected - Database & Sql Blog Articles

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3 Ways to Make Your ELISA Kit Perform Beyond Expectations

1. Reagent Preparation

Proper preparation of reagents is crucial for accurate ELISA results. Always follow the instructions provided with your ELISA kit. Use distilled or deionized water for all steps, including washing, and ensure it's fresh and of high quality. When measuring buffer solutions, use a pH meter for accuracy. If you take reagents from the refrigerator, allow them to reach room temperature before use. Any unused components should be returned to the fridge promptly to maintain their integrity.

2. Washing

Although washing is not a chemical reaction step, it plays a vital role in the success of an ELISA experiment. It helps separate bound and free enzyme-labeled molecules by removing unbound substances and non-specifically adsorbed contaminants. This step ensures that only specific interactions remain, improving the signal-to-noise ratio. Washing is often considered the most critical technique in ELISA, so it must be performed carefully and according to the manufacturer’s guidelines.

There are two common washing methods: immersion and running water. For immersion, after removing the reaction solution, fill the wells with wash buffer, let it sit for 1–2 minutes, then remove the liquid. Repeat this process 3–4 times. For running water, especially for bead-based systems, rinse under tap or distilled water, then immerse in distilled water again. This method is faster and more thorough, particularly when using microtiter plates.

The washing buffer typically contains a non-ionic detergent like Tween 20 (0.05%–0.2%), which helps reduce non-specific binding by disrupting hydrophobic interactions between proteins and the solid phase. However, too much detergent may cause antigens or antibodies to detach, reducing sensitivity.

3. Incubation

Incubation is essential for antigen-antibody binding in ELISA. This step involves allowing the reaction to proceed at a controlled temperature and time. Common incubation temperatures include 37°C, room temperature (20–25°C), and 4°C. Most reactions work best at 37°C, where the binding is efficient and reaches equilibrium within 1–2 hours. Higher temperatures (like 43°C) can speed up the reaction but may affect specificity. Lower temperatures (like 4°C) allow for longer incubation times, often used in radioimmunoassays, but are less common in standard ELISA procedures.

To ensure even heating, use a water bath or a wet box. Avoid stacking plates to prevent uneven temperature distribution. At room temperature, keep the environment stable and within the recommended range. Always monitor the time and temperature closely—accuracy is key to reliable results. If multiple plates are being processed, limit the number handled at once to avoid errors.