Even though the enzyme-linked immunosorbent assay (ELISA) is a common method of measuring the amount of antigen in a sample, it is by no means trouble-free. The many steps involved expose multiple opportunities for something to go awry: immobilizing antigen to the microplate surface, the binding of an enzyme-linked antibody to the antigen, adding enzyme substrate – all separated by numerous washes and blocking incubations. Indeed, even the seemingly innocuous, often tedious, incubations with blocking buffers and wash solutions can take down the entire assay if not done correctly. But reliable troubleshooting solutions can help to solve nearly any ELISA problem. After all, almost any problem you might encounter has probably already been solved by someone else.
At the end of the assay, your ability to read your ELISA and derive meaningful information from it will greatly depend upon the signal-to-noise ratio of the results. Background noise can easily foil your best plans. So keep in mind these helpful tips for reducing background noise in your ELISAs.
Washing is crucial
These tiresome but crucial wash steps are necessary because if any unbound material (such as nonspecifically-bound antibodies, or detection reagent) remains in the microplate wells, it can increase background noise. If necessary, non-specific binding interactions can be discouraged by increasing the salt concentrations in your wash buffer. If background is too high and you suspect that the wash steps were insufficient, you can try doing additional washes, adding detergent or protein to your wash buffer if appropriate, or soaking for a few minutes between washes.
Blocking is also (perhaps more) crucial
The job of a blocking buffer is to bathe the potential binding sites in the plate or microwell – basically any remaining sticky spots – with an irrelevant protein (or proteins) that will bind nonspecifically. This subsequently decreases the opportunity for signal-generating antibodies to bind nonspecifically. You want to make it possible for the antibodies to bind only to the proteins-of-interest for which they are designed to be specific – and to nothing else (which would simply be background). If your background is high and you suspect insufficient blocking, you can try using a higher concentration of blocker, or increasing the blocking time. Hopefully, the blocker will not mask the binding site on the antigen for the antibody.
If you have a problem with persistent background, it might be worth the time investment to optimize the type of block you are using. This may take time, but it will pay off. There are two main types of blocking agents: proteins, and non-ionic detergents. The type you use will depend on many factors, including the surface chemistry of your plates, the antigens adsorbed to them, your antibodies, and your detection reagents. A good blocker should help reduce non-specific binding, but it should also not (or only minimally) react with the antigen, antibodies, or detection reagents.
The most common non-ionic detergent blocker is Tween-20. Detergent blockers are cheap, stable, and useful in removing some non-specific binding during wash steps. But they only work when present, because they can easily be washed away (leaving behind all those unblocked sites). So detergent blockers must be added to all wash solutions, too. Be careful not to use a high concentration (normal concentrations are about 0.01–0.1%), which can reduce specific binding and give you false negatives. Another option is using both protein and non-ionic detergent blockers, where the latter can help to block during wash steps.
Protein blockers, unlike detergents, are permanent. They bind to open spaces and block them, and also stabilize the antigen molecules bound to the microplate. Protein blockers remain after excess blocking buffer is washed away (unlike detergent blockers). Common protein blockers include bovine serum albumin (BSA), non-fat dry milk, normal whole serum, and fish gelatin. For major blocking issues, using whole serum as a blocker can be attractive because the inherent diversity of serum’s components make it effective at blocking many different types of molecular interactions. Whole serum’s disadvantage, though, is that it cross-reacts with Protein A and anti-IgG antibodies, which are often used in detection reactions. A way around this disadvantage is to use whole normal sera from chicken or fish.
Optimize antibody concentrations
Sometimes it’s easier to use a protocol bequeathed to you from the outgoing post-doc, but if your conditions or reagents are slightly different, you may need to change the amounts of antibodies used, for example. Obvious though it may seem, remember that non-specific antibody binding can increase background. To prevent this, don’t use too much primary or secondary antibody – optimize the concentrations needed for your ELISA.
Be diligent with detection reagents
Another point that may seem obvious: don’t use too much detection reagent. Using a concentration that is too high, or diluting it incorrectly, can lead to high background. Don’t overdevelop with the detection reagent – optimize when to use a stop solution, if necessary.
Survey the whole system for optimal results
Look at the components of your ELISA system one-by-one, and ask yourself whether you have optimized each reagent. For example, if you’ve tried different concentrations or blocking times, maybe try a different kind of blocker. Take the plunge and make an experiment out of it – ultimately, optimizing your ELISA reagents will let you reap the rewards of lower background and stronger signal.
The image at the top of this page is from Thermo Scientific Pierce Protein Research Products.