Enzyme-linked immunosorbent Assay (ELISA) kits are powerful tools in the fields of research and diagnostics. Their versatility and precision make them essential for detecting and quantifying specific proteins and molecules. However, even experienced scientists can make errors when using ELISA kits, potentially compromising results. In this article, we will explore ELISA seven common mistakes to avoid when using ELISA kits to ensure the accuracy and reliability of your experiments.
7 Common ELISA Mistakes
Despite how straightforward Elisa Kits can be, it requires much expertise. Even then, errors are not uncommon. Here are 7 common mistakes to avoid when using ELISA Kits.
Plates Stacking
Elisa kits usually have plates on which antigens are immobilized and antibodies combined. This combination is then put in an incubator. The incubators typically have space for multiple plates; however, some days, you might be tempted to stack the plates due to space constraints. It is not a good idea.
Stacking plates during incubation can result in the edge effect. This occurs when outer wells are subjected to conditions different from their nearby inner wells, including varying temperatures. This produces inconsistent results.
Even without stacking, it is advisable to replicate well assays to account for wells impacted by the edge effect. This will enable comparisons to be performed when significant variations appear.
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Pipette Tip Reuse
Pipette tips are disposable pieces that are attached to the tips of pipettes to make dispensing easier. They protect the pipettes from contamination and corrosive substances. Now, while it is unnecessary to exchange pipette tips for every preparation or replicate, it is advisable to do so when transitioning between several samples.
You don’t want contamination to taint the outcomes of your experiment. In general, you should dispose of pipette tips if you cannot guarantee their sterility or suspect any compromise.
Reagent Reuse
Reagents are essential in optimizing the process of antibody detection; hence, they must be of the highest quality and free of interference. These reagents can be expensive or difficult to get, causing people to want to conserve resources by using reagents across different kits.
But doing that is not advisable when using an Elisa kit. This is because each reagent in the kit has been customized for that particular lot. Therefore, reagents from various batches shouldn’t be combined. Furthermore, earlier reagents can deteriorate based on the manufacturing date and storage circumstances.
Combining reagents can adversely affect the ELISA’s capacity for detection, causing inconsistent experiment results.
Drying Plates
After using the plates, it is natural to clean them thoroughly to make them available for use subsequently. When it’s time for use, the plates can sometimes not be dry enough. This sometimes causes you to dry them thoroughly or entirely.
However, according to ELISA protocol, the 96-well plate should be inverted, and any remaining fluid should be tapped onto paper towels or other absorbent material. While ensuring all remaining moisture is gone, you don’t want the plate to dry fully. This is because it can cause the active substances on the plate to become inactive, impairing the assay’s effectiveness.
Reagent Preservation
Reagents need to be stored appropriately and at specific temperatures. However, they should be brought to room temperature when they are to be used unless otherwise stated. It is also crucial to keep the reagents’ exposure to freeze-thaw cycles to a minimum.
This is because each cycle can reduce the efficiency of the reagents. When you receive the reagents, single-use aliquots should be made to keep them in optimal shape. Doing this adds one more step to ensuring repeatable and consistent results.
Incubation Time
Incubation time refers to how long your preparations should stay in an incubator. This period allows for the development of detectable signals and is essential in evaluating antibody levels. Various analytes require various incubation periods and temperatures, which must be adhered to.
Not adhering to them can lead to suboptimal incubation and can cause underreporting. Extended incubation time can, however, generate overly high signals and give you false results. For the suggested incubation times, consult the accompanying data sheet.
This also applies to incubation temperatures because incubating at optimal temperatures reduces the risk of false results because of nonspecific antibody binding.
Sample Preparation
A myriad of problems can arise during the sample preparation process. There can be an intricate problem with the chosen method. This is because methods are specific to different tests. Assay condition, sample quality, and reagent quality should also be closely monitored as they are places where errors can occur.
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Choosing the Right ELISA Kit
Selecting the appropriate ELISA kit is the first critical step in ensuring the success of your experiment. There are various types of ELISA assays, including Direct, Indirect, Sandwich, and Competitive ELISAs. The choice depends on the specific analyte you aim to detect. Using the wrong type can lead to inaccurate results. Additionally, it is essential to ensure that the kit you choose is compatible with the target analyte.
Proper Storage and Handling
Once you’ve selected the right ELISA kit, proper storage and handling are vital to maintaining the integrity of the reagents. ELISA kits typically contain sensitive antibodies and enzymes, which can become inactive if exposed to unfavorable conditions. Always follow the manufacturer’s instructions for storage, including temperature and humidity considerations. Avoid contamination by keeping the reagents and samples separate, and never return unused reagents to the original container to prevent cross-contamination.
Sample Collection and Preparation
The quality of the samples used in an ELISA assay directly impacts the accuracy of the results. When collecting samples, ensure that they are of high quality, free from contaminants, and properly stored. Sample handling is crucial, as proteins and molecules can degrade over time or due to temperature fluctuations. Follow best practices for sample handling, such as using protease inhibitors, and process samples promptly to minimize degradation.
Calibrating and Standardizing
Calibration and standardization are essential to ensure that ELISA results are accurate and comparable. Calibration standards provide reference points for the concentration of the target analyte. Proper dilution and concentration techniques must be followed to ensure that the standards accurately reflect the concentration of the samples. Failing to calibrate and standardize can lead to unreliable results and hinder the ability to compare data between experiments.
Assay Controls and Quality Assurance
Proper controls and quality assurance measures are key to the success of an ELISA experiment. Positive and negative controls help validate the accuracy of the assay. A positive control should yield a known, detectable signal, while a negative control should yield no signal. Without these controls, it can be challenging to determine if the assay is functioning correctly. In addition, regular validation and troubleshooting are essential to address unexpected issues that may arise during the assay.
Data Interpretation and Reporting
Accurate data interpretation is the final step in obtaining meaningful results from an ELISA experiment. Ensure that the data is analyzed correctly, using the appropriate software or techniques. Interpretation should consider factors such as the standard curve, background correction, and the appropriate statistical analysis. Presenting results clearly and effectively is also crucial. Figures and graphs should be labeled and explained, and the methods used should be transparent.
Conclusion
In conclusion, ELISA kits are valuable tools in research and diagnostics, but their accuracy and reliability depend on proper handling and technique. By avoiding the ELSIA common mistakes discussed in this article, scientists can enhance the precision and quality of their ELISA experiments. From choosing the right kit to interpreting the results, each step plays a crucial role in ensuring the validity of the data collected. As researchers continue to push the boundaries of science, attention to detail and adherence to best practices in ELISA experiments will remain essential for success.
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