When conducting ELISA experiments, the choice of reagents plays a crucial role in ensuring accurate and reliable results. Different manufacturers may produce reagents with varying levels of sensitivity and specificity. Using low-quality or substandard reagents can lead to false positives or false negatives, which can significantly impact the validity of your findings. Therefore, selecting high-quality ELISA kits is essential for achieving consistent and precise outcomes.
This article provides a comprehensive guide on common issues that may arise during ELISA experiments and offers practical solutions to help you avoid them. Whether you're a beginner or an experienced researcher, understanding these challenges can greatly improve your experimental success rate. Read on to learn more about how to optimize your ELISA process.
| Steps | Possible Causes | Solutions |
| Reagent Selection | Selecting low-quality reagents may affect test accuracy. | Choose high-quality reagents from reputable suppliers, follow instructions carefully, and allow reagents to reach room temperature before use (about 30–60 minutes). |
| Loading | Improper sample handling, delayed incubation, or enzyme spillage may occur. | Ensure proper sample preparation: for serum, let blood clot for 1–2 hours then centrifuge at 3000 rpm for 15 minutes; for plasma, use anticoagulant tubes and invert after collection. Store samples appropriately and load them into the incubator promptly. After adding the enzyme reagent, gently blot the plate with filter paper to remove excess liquid. |
| Incubation | Incomplete coverage or extended incubation time may cause non-specific binding. | Always cover the plate during incubation and strictly adhere to recommended incubation times to prevent over-binding. |
| Washing | Manual washing may cause cross-contamination, while improper washing machines may not clean effectively. | Ensure complete filling of wash buffer in all wells and check for clogs. Pat the plate dry with clean absorbent paper after washing. Use multiple washers if available to reduce waiting time. |
| Color Development | Expired developer or improper addition may result in inaccurate readings. | Prepare the developer just before use and avoid using expired solutions. Ensure no overflow occurs when adding the reagent. Keep A and B components away from metal surfaces to prevent contamination. |
| Termination | Bubbles in the stop solution may increase false positives. | Add the stop solution carefully to avoid air bubbles. |
| Reading | Residue on the bottom of the plate may interfere with readings. | Clean the plate thoroughly before reading to ensure accurate optical density measurements. |
| Overall Process | Exposure to contaminants or lack of standardization may affect results. | Avoid exposure to hypochlorous acid and consider automating the ELISA process for greater consistency. Implement internal quality control and participate in external quality assessments to maintain high standards. |
In addition to using high-quality reagents, it’s important to strictly follow each step of the procedure. Maintaining good laboratory practices, such as performing internal quality control and participating in inter-laboratory comparisons, will further enhance the reliability of your results. Many laboratories in China now use automated microplate readers, which play a key role in standardizing ELISA testing and improving overall testing quality.
We offer premium ELISA kits at competitive prices. If you need assistance or have questions, feel free to contact us via phone, QQ, or email. Our ELISA kits are available in 96T and 48T formats to suit your testing needs. Don’t miss out—reach out today!
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