Have you ever heard about ELISA? Such a test is conducted to diagnose various diseases, such as HIV, syphilis, and rotavirus, as well as to measure antibodies in the blood of individuals. It has to be run successfully for the outcome to be consistent and credible.
Nevertheless, it often happens for issues to occur and influence the outcome, like problems with the standard solution, detection systems, or difficulties in obtaining a signal.
Have a look at some of the most prevalent problems with ELISA and their corresponding solutions.
Standard solution issues
During the process of setting up a standard curve, users might encounter issues with the standard solution. In such scenarios, the solution has either not been diluted correctly or has been improperly reconstituted. In the first case, you should always confirm that dilutions are made in a correct manner, whereas in the second case, you’re advised to spin the vial briefly prior to opening it. You should also inspect for any undissolved material following reconstituting.
Another potential cause might be a degraded standard, which can be avoided if the standard is stored and handled as recommended. A pipetting error might result in such an issue, which is why you’re expected to use calibrated pipettes and the correct pipetting technique. Find out how to perform pipette calibration.
Inconsistent results and high CV (coefficient of variation)
Another potential problem you might encounter is getting inconsistent results and a high coefficient of variation. The CV refers to the amount of signal variation between runs, expressed in the form of a percentage. It should always be lower than twenty percent. The potential causes for this issue involve bubbles in wells, inadequately washed wells, inconsistent pipetting, edge effects, etc.
If bubbles in wells are the source of the problem, next time, you should be certain there are no bubbles before reading the plate. Bubbles can be removed with the assistance of pipetting up and down in a gentle manner. When wells aren’t washed thoroughly, you have to check whether the plate washer ports are unobstructed.
Inconsistent pipetting is another reason why the results might be inconsistent. You need to use calibrated pipettes and the right technique, as well as make sure all reagents are mixed correctly. Conversely, edge effects tend to happen in ELISA when wells are exposed to even slight temperature variations. The edge or outer wells are the first to provide a response to any environmental changes, which cause them to evaporate much sooner than the inner wells.
Sometimes, the edge effect is visible just by looking at the plate. When the buffer level is lower on the edge wells, it indicates that a portion of the buffer dried out. You can prevent such drying by covering the walls with tape or sealing film. There are various Elisa Troubleshooting Tips online, providing assistance in some of the most common troubleshooting areas. When coping with edge effects, the plates aren’t supposed to be used right out of the fridge, given the inner wells need some time to adapt to room temperature.
Problems with obtaining a signal
Another issue that you might come up with is having difficulties in obtaining a signal. The inability to obtain a signal might occur due to poor absorption of the antibody to the plate, absence of the protein of interest, insufficient antibody bound to the protein, etc. When the antibody absorbs insufficiently to the plate, you can increase the plate’s absorption by pre-treating the wells.
In case the protein of interest is absent in the sample, you should conduct a positive control and consult scientific literature to check whether the protein should be present. When an insufficient antibody is bound to the protein, you need to add a higher primary antibody concentration and prolong the incubation period of the sample.
Detection system problems
Detection system issues are also common with ELISA. They might occur when detection is insufficiently sensitive or improper filter settings are applied for detection. If detection isn’t sensitive enough, you should try switching to a system with higher sensitivity, such as from colorimetric to fluorescence. Perhaps, you might need a switch from direct to indirect detection.
If incorrect filter settings are used, you should check the condition of the plate reader. This instrument is expected to be set to the adequate absorbance wavelength or emission wavelength. Detection system issues can be brought about by slow colorimetric reaction development.
Certain colorimetric reactions develop slowly, meaning they might not be complete if the plate is read early. See this URL, https://en.wikipedia.org/wiki/Colorimetry_(chemical_method), to find out more about colorimetry. Consequently, part of the signal might be missed.
To sum up
Early detection of these problems is crucial for them to be addressed quickly and properly.
Otherwise, the outcome of ELISA won’t meet the expectations!