The Enzyme-Linked Immunosorbent Assay, or ELISA, is a cornerstone of biotechnology – it plays a critical role in scientific research, food science, and medical diagnostics. We’ve written before about using the ELISA to detect West Nile and SARS-CoV-2, and have a free Edvotek at Home ELISA activity for you to share with your students. Today, we are going to discuss ways to avoid common errors and master the ELISA in your classroom.
Preparing your samples
Every ELISA will have unique components that are designed to detect a specific antigen/antibody, depending on the scenario that is being run. However, almost every ELISA will feature a combination of antigen, primary antibody, secondary antibody, and substrate. It is vital that the proper protocol is followed to prepare the reagents and perform the activity, which means that the first step should always be to check that the literature you are using matched the reagents you have on hand (check the paper sheet that comes with the reagents!). Almost all ELISA components that Edvotek provides have been lyophilized (freeze-dried) and will need to be rehydrated prior to use – follow the instructions in the literature and make sure that everything has been fully resuspended in the buffer before dispensing. The lyophilized components can occasionally stick to the glass vial, so add your buffer, let it sit for a minute or two, and then pipet up-and-down a few times to ensure everything is dissolved.
The reagents should be rehydrated as close to the start of the experiment as possible, preferably within the same week that they will be used. If you would like to aliquot reagents ahead of time it is best to keep everything COLD – use ice to keep the samples and tubes cold while you aliquot and then store in the refrigerator until needed. Never freeze the ELISA components, this will destroy the activity. Finally, make sure that you are using the correct buffer – most ELISA experiments will have 2 or 3 different buffers that are specific to certain steps!
Wash the wells the best way possible
Washing the wells is one of the key steps for every ELISA. The strips can be dumped into a sink, waste beaker, or onto a paper towel, making sure to not spill samples from one well to another. After dumping out the components the ELISA strips should be tapped onto a paper towel to remove all remaining sample. Once the tubes have been fully emptied, they should be CAREFULLY filled with wash buffer, taking care that it does not spill into neighboring wells. It’s okay to leave a little space at the top of each well, but ideally the wells will be mostly filled with buffer.
Knowing when to stop
The ELISA will normally take a full 45-50 minute class period to complete, but can occasionally take longer. It is important to plan ahead if you think that you will need to divide the experiment between multiple days. The experiment can be paused during any of the wash steps, making it fairly flexible. Simply wash the strips like normal, then refill each well with wash buffer and store in the refrigerator until needed – it’s best to return to the experiment within 24 hours if possible, but most experiments should be fine for 48-72 hours. To prevent evaporation from the samples we recommend covering with plastic wrap or aluminum foil, or placing the ELISA strips into a plastic bag.
At the conclusion of the ELISA you should see a vibrant color change across well, indicating the presence or absence of antigen/antibody in the starting samples. The samples will continue to darken over time, including in wells that are negative. Because of this, it is important to analyze the samples before they have a chance to saturate. The good news is that students can take a picture of their samples at any point – snap a photo after 2 minutes, then again after 4 minutes, and so on. Some quantitative ELISA experiments will contain specific instructions on stopping the reaction, including the addition of a stop solution. These experiments should be allowed to develop until the darkest wells (with the highest concentration of antigen) are no longer changing color, which indicates that the reaction has saturated.
Analyzing your results
Each ELISA has a unique pattern of expected results, depending on the scenario that you are running or the samples you are analyzing. However, it’s possible that the students will see something unexpected. The most commonly seen erroneous results include fully negative (no color change) or fully positive (everything changes color) samples, or a random pattern of positive and negative samples.
A fully negative strip of samples. This is most often caused by incorrect dilution of the reagents, use of the wrong dilution buffer, not fully suspending the samples before aliquoting, or by students skipping a step. It is also possible that the reaction has not progressed for enough time – allow the samples to incubate for a few more minutes to see if a color change occurs.
A fully positive strip of samples. In this strip, every well changed color, including the negative controls. These results often occur from improper washing (not fully removing samples or incomplete washes), adding reagents in the wrong order, or cross-contamination from samples spilling into neighboring wells. This can also occur if the reaction is allowed to incubate for too long at the end of the experiment, allowing the color-change reaction to saturate the wells.
Like every lab technique, the ELISA takes attention to detail and proper laboratory skills. One of the best ways to prepare students is by having them watch a video overview of the procedure.
For more information on the ELISA visit our ELISA Learning Center, where you’ll find links to presentations, activities, and lesson plans!