ELISA are immunoassays widely used in biomarker detection and validation. They have been used in research and clinical settings for more than 40 years, and they allow to quantify, in a simple way, thousands of biomarkers. New ELISAs are being developed every day, either for new biomarkers or new species. They can also be used to detect contaminants in food, water or industrial processes.
Technology-wise, there are several types of ELISAs, depending on whether they are based on double (or sandwich) antibody detection, direct detection, competitive detection, etc. It is not the scope of this post to enter into details about the different formats that an ELISA can have, but if you think that you need this info, feel free to suggest that I do a post on that!
Nevertheless, the aim of this post is to give some hints on how to reach the best result for your ELISA tests, based on some troubleshooting experience with researchers and clinicians all over Europe.
Hint # 1 – everything counts
First, check that you have all reagents and your samples on hand.
Give them a spin to get all the volume down. And then follow the protocol, adding all that needs to be added. Believe it or not, in some (exceptional) cases, some ELISAs don’t work… because either no sample or one of the reagents wasn’t added. Don’t take anything for granted. Check every step!
Hint # 2 – keep cool
All reagents should be stored at the recommended temperatures. And if you intend to use your ELISA in several rounds, make sure that you aliquot to avoid freeze/thaw cycles. Freeze/thaw especially affects the standards and other reagents (e.g. detection antibody). Dramatically. Trust me.
Also, freeze/thaw may have an influence on the samples themselves. As a rule of thumb, never freeze/thaw anything. What applies to food in your fridge also applies to biological reagents and samples.
Hint # 3 – be gentle with the standards
When preparing your standards, it is very critical to spin down the vial first (yes, I have already mentioned this, but let me insist). The powder may drop off from the cap when opening it if you do not spin down.
Be sure to dissolve the powder thoroughly when reconstituting. After adding Assay Diluent to the vial, invert the tube a few times, then flick the tube a few times, and then spin it down; repeat this procedure 3-4 times. This is a very effective technique for thoroughly mixing the standard without too much mechanical force (do not vortex!).
Also, keep the standard dilutions on ice during preparation (but the ELISA procedure should be done at room temperature). And discard the working standard dilutions after use – they do not store well.
Hint # 4 – the dark side
Some of the reagents in an ELISA kit are light-sensitive. TMB solution, for example.
Make sure that you store it in the dark. Also, keep the ELISA plate in the dark while the color develops. This is not mandatory, but it really helps most of the times to get a better development of the signal and avoid low ODs.
Hint # 5 – detecting the undetectable
Make sure that you choose the right ELISA. Not all biomarkers are present at the same concentration in all sample types. Serum is usually the best sample that you can use for ELISAs. Plasma is not too bad, as long as it is not hemolyzed (and if it is, be ready for high values due to background noise). Urine is quite fine, but remember recovery is lower, so you will be losing sensitivity. Other samples such as saliva, BAL and CSF can be tricky, not only due to the low amount of biomarkers usually present there, but also due to inhibitory effects. It’s a question of case-by-case, so don’t use your most valuable, cherished clinical samples to run tests!
Also, have a look at the literature to check the usual levels of the biomarker of your interest in your specific experimental model or disease. And choose the ELISA with the right detection range. If you see that, let’s say, IL-6 is usually at 6 ng / ml in the disease you are studying, make sure that the ELISA that you choose has this concentration in the linear range of the standard curve (as a rule of thumb, the linear range starts at point 2-3 in the curve). You might also have at look at a previous post describing criteria used to select the most appropriate ELISA kit to detect Human Cardiotrophin-1.
And always remember that if you choose the right ELISA, with the right detection range, and you just don’t detect the biomarker in your samples, it might well be that it is not there. As simple as that.
In any case, if you’re not sure, and have some samples to test, there are trial programs to allow you to test, risk-free!
Hint # 6 – ask the experts
If you followed all these points and still had a problem with the ELISA kit, check with the manufacturer. Fortunately, we seldom get incidents with ELISA kits, but when we do, we have some points to help improve the results, sometimes dramatically. Some examples:
a) Try incubating overnight at 4 ºC to increase OD (and remember hint #4).
b) Increase concentration of HRP by 1.5 – 2 fold.
c) Increase time following TMB solution addition to allow for increased colour development.
d) Older plate readers can cause lower OD. Rare, but it happens.
e) If you want to check whether HRP:TMB is not the issue, spike some HRP (2 – 5 ul) into a clean tube, add some TMB (5 ul) and see if there is a blue colour change. If the colour is weak…contact the manufacturer.
What about you?
I’d like to thank Jarad and Daniel (Raybiotech) for their technical support on writing this post! May be you have also other tips you would like to share!
To do so, please leave your comments below!