7 Tips for Outstanding ELISA Evaluation
Enzyme-linked immunosorbent assay, also known as ELISA, is a technique used for detecting and qualifying hormones, antibodies, peptides, and proteins. It has numerous applications, being used in the diagnosis of HIV, syphilis, Lyme disease, and toxoplasmosis, among others.
ELISA test kits are highly useful screening tools that can make further testing much easier. But being extremely sensitive, you need to be very careful with how you perform ELISA evaluations.
To receive consistent, accurate, and reproducible results, take a look at the following tips for outstanding ELISA evaluation.
Choose the Right ELISA Test Kit
There are four different types of ELISA:
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
The type of ELISA you should choose depends on your reagents and the required output. If you need fast results, simple direct or indirect ELISA will suffice. If you need higher flexibility and sensitivity, then the more complex sandwich or competitive ELISA would be preferred.
Use the Right ELISA Plates
It’s of utmost importance to use plates explicitly designed for ELISA. This will ensure consistency and minimize the risk of the occurrence of common ELISA errors.
You’ll need 96-well, flat-bottomed plates. It’s often an excellent choice to test out different plates from different manufacturers and check for any variability between them.
Follow Simple Steps for Improved Efficiency
Before commencing with the test, it’s best to get organized. Write down the step-by-step instructions; this will optimize the process and ensure greater efficiency.
Make the job easier for yourself by using a contrasting background, such as a dark piece of paper placed under the ELISA plate. This will improve the visibility of all your samples.
Lastly, place your samples in the order that you’ll use them. This will minimize the risk of error and help you stay on top of the ELISA evaluation.
Avoid a Common Mistake
Although it’s not always necessary to change your pipette tips for every single addition to the assay, it’s still in your best interest to do so.
It will prevent cross-contamination between the different wells and ensure that you have a low coefficient of variability (%CV).
Know the Common ELISA Errors
Being so sensitive, ELISAs are sometimes prone to errors. The most common mistakes you’ll encounter are the edge effect and the hook effect.
The edge effect usually occurs when ELISA multilevel plates have inconsistencies in the production (which is why testing out different plates is so crucial). This error is common if you stack plates as well, and you’ll receive different results in the outer wells.
The hook effect is common with high levels of antigen in your samples, and you’ll see a lower signal than what you’d expect.
Have Duplicates
To handle the common ELISA errors and improve the accuracy of your evaluation, it’s crucial to have duplicates for every single point of your assay. This means duplicate background, sample wells, standard wells, etc.
Be Consistent
After you’ve optimized the assay, it’s crucial to keep all conditions unchanged. ELISA is highly sensitive, and even the smallest of changes in the antigen and antibody concentrations, washing times, buffer composition, incubation times, etc. will affect the end results.
Always follow the same steps, and always follow the guidelines that come with your ELISA test kits.
In Conclusion
Following these tips will ensure outstanding ELISA evaluation every single time. They all seem minor, so they’re easily forgotten, but they’re crucial nonetheless and will provide you with consistent, accurate results.
