ELISA has been around for over five decades, and it is still the preferred method for many researchers when detecting and quantifying antibodies in serum or plasma samples. The steps of ELISA are relatively simple: capture antibody with antigen, incubate, wash away unbound material, add enzyme-linked secondary antibody, incubate, wash away unbound material, add substrate, and develop a color reaction.
The process may sound easy, but it can fail at any of these steps. The key to success is following the protocol to the letter, being mindful of potential pitfalls, and knowing when to troubleshoot. Here are some tips for getting the best results with your ELISA experiments.
Choose the Appropriate Kit
Various assay testing kits perform best for a particular type of protein. Their surfaces, reagents, and detection systems target a specific antibody or antigen. After deciding on the proteins, find an ELISA kit designed for them.
The kit’s insert will list the proteins and antibodies that work with the reagents. Some manufacturers also have online tools to help you choose the best one. If in doubt, ask customer service for assistance. Also, understand if your antibodies are monoclonal or polyclonal. That will determine whether you need a capture or sandwich ELISA.
Ascertain the Reproducibility of Your Antibodies
Reproducibility is the ability of an assay to give the same results each time you do the test. The best ELISA outcomes come from antibodies that perform reproducibly in the assay. A viable ELISA will have a low coefficient of variation (CV). Ascertain reproducibility when working with custom antibody services.
To confirm that your antibodies are fit for ELISA, test them against various concentrations of the protein they are directed against. It will give you a standard curve and help you determine the sensitivity and dynamic range of the antibodies. It will also help you to optimize your ELISA conditions, such as incubation times and concentrations of reagents.
Employ Good Sample Handling Practices
To get good ELISA results, you need to start with high-quality samples. Collect blood samples from fasting subjects in tubes that contain an anticoagulant. Store them at the correct temperature and use them within a few days.
If working with plasma or serum, centrifuge the blood samples as soon as possible to separate the cells from the clear liquid. For long-term storage, freeze the samples in aliquots at -20 °C. Avoid freezing and thawing more than three times, as this can damage the proteins.
Not every protein will give the same results when diluted to different concentrations. To find the optimal dilution, start with a range of concentrations and use two-fold serial dilutions. Then, plot the optical density (OD) readings against the protein concentration. The results will form a sigmoidal curve.
The point at which the curve transitions from the linear to the non-linear range is the optimal concentration for ELISA. At this point, you will get the most accurate results with the least amount of variability. When you have found the optimal protein concentration, make a two-fold dilution and use that going forward.
Follow the Manufacturer’s Protocol
Each ELISA manufacturer will have a specific protocol for their kit. That will detail how to prepare the reagents, incubate the samples, and develop the color reaction. For instance, the test kit may recommend adding the primary antibody first, followed by the enzyme-linked secondary antibody.
Follow the protocol exactly as described. Do not shortcut the incubation times or change the order of reagent addition, as this can lead to false-positive or false-negative. Also, never mix reagents from various vendors. Each reagent works with the others in the kit, and substitutions can lead to inaccurate results.
Optimize Incubation Conditions
The incubation is crucial in ELISA, as it allows the antibodies to bind to their targets. Your goal is to optimize it for maximum binding with the least amount of nonspecific binding.
The first step is to determine the optimal antibody concentration. Test a range of concentrations and look at the resulting standard curve. The next step is to optimize the incubation through trial and error. Start with the recommended incubation time and temperature, then adjust as needed. Go in this direction if your results are too variable or have a lot of nonspecific binding.
Incubation can also vary according to sensitivity. Lower sensitivity ELISA may require longer incubation time to allow for more antibody binding. Higher sensitivity ELISA may need shorter incubation periods to avoid signal saturation.
ELISA is a versatile and sensitive tool for detecting proteins, but you must employ viable techniques to get the best results. Use purified antibodies specific to your target protein and dilute them to the optimal concentration. Collect high-quality samples and handle them carefully to avoid damaging the proteins. Also, follow the manufacturer’s protocol for the kit you are using and optimize the incubation conditions for maximum binding with the least amount of nonspecific binding.