The enzyme-linked immunosorbent assay or ELISA is one of the most common antigen detection and antigen measurement process. This, however, does not mean it is a hassle-free process. There are so many steps involved, like immobilizing the antigen, binding the antibody to it, adding the enzyme substrate, etc. that a number of opportunities come up where a simple error could compromise the entire test. Between each of these steps, too, you are expected to subject the apparatus to incubations and washes with blocking buffers or wash solutions. Even these simple steps could mess up the entire assay if not performed correctly.
Finally, the result’s accuracy would depend on the signal to noise ratio. Background noise is a very potent factor in tests like these, and could affect your results to a great extent. Hence, steps must be taken to mitigate these risks.
Always Remember to Wash
Washing steps seem tiresome and frivolous, but they are perhaps the most important steps of the lot. Any unbound material like detection reagent or non-specifically bound antibodies would increase background noise if allowed to remain in the microplate wells and hence must be washed off. By increasing the salt concentration in the wash buffer, you can also discourage non-specific binding interactions. If your results indicate a high amount of background noise, try doing more washes or adding protein and detergent to your buffer. Another thing you can try is to soak the well for a few minutes between consecutive washes.
Blocking is Vital as Well
A blocking buffer washes all potential sites of binding in the micro-well or plate; these are all the sticky spots with irrelevant non-specific binding proteins. By doing this you can successfully reduce the opportunities for non-specific binding by signal-generating antibodies so that the only antibodies that are binding are the ones binding to the specific proteins of interest, and not the background. Higher concentration of blocker or increased blocking time is effective methods to counter insufficient blocking and to lower background noise. You will have to be careful not to mask the real binding sites on the antigen for the concerned antibody.
Types of blockers: Also understand that it may make sense to optimize your block even if it takes more time. The two fundamental types of blocking agents are non-ionic detergents and proteins. Several factors, ranging from the surface chemistry of the plates you use and the detection reagents to the antigens and antibodies, are responsible for determining the type of block you should use.
Detergent blockers- Detergent blockers are fairly stable, inexpensive and effective in removing non-specific binding. Tween-20 is the most popular non-ionic detergent binder. That said the detergent can easily be washed away before it has completed its work. Hence, they must be added to your wash solutions as well, around 0.01 to 0.1% as a higher concentration could return false negatives.
Protein Blockers- Protein blockers, unlike their detergent counterparts, are permanent in nature meaning they find the open spaces, bind to them and then block them and further stabilize the bound antigen molecules. They remain in the apparatus even after the excess blocking buffer has been washed away. Some of the more common blockers are non-fat dry milk, BSA or bovine serum albumin, fish gelatin and normal whole serum. Whole serum makes sense for major blocking issues due to the diversity in components. The disadvantage with whole serum is that it cross-reacts with anti-IgG antibodies and Protein A, which are used for some types of detection. A simple workaround for this is to use normal sera from fish and chicken. You could also use a combination of non-ionic detergents and protein blockers.
Do not Overuse Detection Reagents
Never use too much detection reagent. If your concentration is very high or you haven’t diluted it properly, you end up with high background. Don’t overdevelop with the reagent and optimize the use of a stop solution if needed.
Optimize the Concentrations of Antibodies
While it is always easy to use the standard protocol or follow instructions from a post-doc, it is definitely important for you to modify amounts of antibodies used if the reagents and conditions vary from those referred to in the instructions. You must remember that non-specific antibody binding increases background, so you mustn’t use secondary or primary antibodies excessively. Tinker around with concentrations till you reach the optimum.
Constantly Survey the System
Inspect each ELISA system component one-by-one and try to figure out if you have optimized every reagent, or if there is any scope for improvements like different blocking times, concentrations or blockers. After this, you are all set to conduct your experiment, with the assurance that you have reduced background as far as possible and will now get a strong signal.
By following these simple guidelines, you will to a large extent be able to assure yourself that the test results will be sufficiently accurate.
Author bio: Dr. Richard Giles has been at the forefront of HIV research at a leading pharmaceutical company. He is especially well-known for his path-breaking research on identification of the GABRB3 antibody.