How to get the most out of your ELISA

How to get the most out of your ELISA

ELISA (enzyme-linked immunosorbent assay) is a common test for detecting and quantifying proteins, particularly antibodies. The ability of ELISA to quantify proteins is the reason you might choose this technique over qualitative methods, such as western blotting.

The (very) basic steps in a standard ELISA are:
1) Antigens from the sample are attached to a solid surface.
2) An antibody that will bind to the antigen is added. The antibody is linked to an enzyme.
3) The enzyme’s substrate is added, causing a reaction that gives a detectable result (such as a colour change.)

ELISAs are relatively straightforward and can be inexpensive to carry out when carried out effectively, and can also be automated. As such, they are a commonly used technique and will be familiar to many researchers. However, as with any common practice, there is a risk of complacency through familiarity. In any laboratory method, there are things that can go wrong – and a number of ways to make sure you get the best possible results.

It is worth noting that a range of sandwich and competitive ELISA kits is available (at St John’s Laboratory, we have over 500 ready-to-go ELISA kits, including sandwich and competitive ELISAs, available here.) Your first step could be to check whether one of these kits meets your needs.

Whether you plan to use a kit or develop your own ELISA, here are a few tips to help you get the most out of your method.

1. Choose your antibody carefully

In an ELISA, detection of the target protein depends on the specific antibody-antigen interaction. The more specific the binding, the better your results should be. It is therefore important to take care when choosing your antibody.

It is also important to choose an antibody with high affinity for the antigen; this should help to reduce non-specific binding.

2. Check your antibody concentration

If you are developing and optimizing your own ELISA, you will need to carry out antibody titres to identify the concentration that produces the highest level of specific binding. If you are using a secondary, or detection, antibody, that can also be titred with the capture antibody to find the best signal-to-noise ratio.

3. Be consistent - it's about accuracy and precision

With any assay, consistency and standardization of the procedure is extremely important. A ready-to-go ELISA kit should have been standardized and optimized for lot-to-lot consistency, as well as for specificity and sensitivity, etc. However, it is still important to aim for consistency in your use of the kits. Checking and following the recommended procedure each time you use a kit can help with this. Also, avoid mixing components from different kits.

Whether you are using a kit or developing your own ELISA, keeping conditions such as temperature and humidity constant, and following the same protocol each time, can help to improve the reproducibility of your results.

If your results are inconsistent from one assay to the next, first check your protocol. Fluctuating temperatures during incubation, insufficient washing, or incorrect dilutions could also produce inconsistent results.

4. Wash carefully with buffer

Between each step of an ELISA, the plate is washed to remove any non-specifically bound proteins. This washing is one of the factors that makes ELISA such a powerful tool. Poor washing, however, can cause problems ranging from too much background to inconsistent replicates.

If you are using a kit, make sure to follow the manufacturer’s washing guidelines, including on how many times the process should be repeated. If the ELISA is your own, don’t neglect the washing step when developing your protocol. Choosing an appropriate washing buffer from the many available is important; the right choice can help to enhance signal and reduce background.

These are just a few of the factors that could help you to get the most out of your ELISA. In addition to our ELISA kits, our 30,000-strong catalogue includes many antibodies suitable for use in ELISA. Browse our catalogue to check out the various applications and the validation status of our antibodies.