ELISA kit experiments are widely used due to their high sensitivity and strong specificity. However, every step in the procedure can significantly impact the final results. If not carefully managed, issues such as incomplete color development or uneven plate staining may occur. To help researchers avoid common pitfalls, our company has compiled a detailed guide on potential problems during each stage of the process and provided practical solutions to improve experimental outcomes.
1. Selecting Reagents
It's crucial to use high-quality reagents and follow the manufacturer’s instructions precisely. Before starting, allow all reagents to reach room temperature for 30–60 minutes to ensure optimal performance.
2. Sample Loading
Possible issues include poor separation of serum or plasma, excessive sample volume leading to long waiting times before incubation, or enzyme spillage. To prevent this:
- For serum: let blood clot naturally for 1–2 hours, then centrifuge at 3000 rpm for 15 minutes.
- For plasma: use anticoagulant tubes and invert them 5–10 times after collection. Centrifuge at 3000 rpm for 15 minutes. Store at 2–8°C if testing within a few days, or at -20°C for long-term storage.
- Load samples promptly and dry the plate with blotting paper after adding the enzyme reagent.
- Use automated systems like FAME or AT when handling large batches.
3. Incubation
Improper incubation can lead to evaporation or non-specific binding. Always cover the plate and strictly follow the recommended incubation time.
4. Washing the Plate
Manual washing can cause cross-contamination between wells. For semi-automatic washers, ensure sufficient washing buffer is used, no clogs exist, and the plate is washed thoroughly. Avoid long delays between steps.
5. Color Development
Using expired developer or leaving it too long can affect results. Prepare the developer just before use, avoid exposing it to light, and keep it away from metal tools. TMB developer should be handled carefully as it is not visible to the naked eye.
6. Stopping the Reaction
Bubbles in the stop solution can cause false positives. Add the stop solution slowly and evenly to avoid air bubbles.
7. Reading the Plate
Ensure the plate is clean before reading. Avoid contact with hypochlorous acid and consider using automated readers for more consistent results.
In practice, attention to detail and strict adherence to protocols are essential. Performing internal quality control and participating in inter-laboratory assessments can further enhance accuracy. With the increasing use of automated microplate readers in China, standardization of ELISA procedures has become more achievable, improving overall testing quality.
I hope this information helps you in your ELISA experiments. Pay close attention to every detail, and always double-check your work to ensure reliable and accurate results.
This content is derived from the advanced enzyme-linked immunosorbent technology developed by Shanghai Jinma Experimental Equipment Co., Ltd. It incorporates the expertise of domestic and international specialists. By leveraging cutting-edge technology and equipment, we aim to maintain high-quality standards while reducing costs, helping researchers save on experimental expenses and contributing to national scientific progress.
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