How to do an ELISA (or A day in the Fever Lab)

Arista Botha


An example of a 96-well ELISA plate. Photo by Arista Botha


When you are writing up your PhD protocol, your experiments all seem simple. Then, when it gets to actually carrying out the experiment, things are often a lot more complicated than what they appeared like on paper. For example, my methods description was strewn with all of the blood chemistry tests one could possibly think of. Once my protocol and my ethics were approved, I had to start making it happen. How hard could it be?


As it turns out, finding an ELISA kit for any animal other than humans, rats or mice is tricky, so I ended up sending about twenty emails back and forth to various companies. Finally I found a company that could send me the right ELISA kit. The second surprise came when I saw what ELISA kits cost! I never knew that such a small thing could be so expensive. Luckily, my supervisor had a grant that was just about the right amount to cover my ELISA kits. So I had a product, a supplier, a quote and a grant. Satisfied, I placed my order. I was so excited when, a few days later, my kits were delivered. Like a little kid opening a Christmas present, I unpacked my kits, marked them clearly with my name, and stored them neatly into the fridge.


I stood back and thought: Now what!


Panic started to set in. During my Master’s degree I had spent most of my time in the field, tracking my study animals and doing behavioural observations. The result being that I have not spent any time in a laboratory since my undergraduate degree! How did I get myself into this?


Luckily Tanya and Lois from the Fever Lab were doing ELISA’s for their research, and they agreed that I could tag along and they would teach me how to do an ELISA. So I joined the Fever Lab for a day.


First off, I have to say that the Physiology Department have the most amazing equipment in Monica’s assay lab! To start off with, Tanya used an ultrasonic homogenizer (also called a sonicator) to prepare rat brain tissue before performing an ELISA for detecting rat IL-1β protein. A sonicator is similar in purpose to a homogeniser (or the blender in your kitchen) except that a sonicator uses ultrasonic sound waves to disintegrate the tissue. ULTRA-SONIC SOUND WAVES! That sounds like something from a science fiction movie…



An ultrasonic liquid processor (sonicator) is used in our lab to sonicate the samples. Photo by Arista Botha


And then, they have this “super-pipette”, which we call the “Pipette-man”! The first part of doing an ELISA is pipetting your sample and other solutions provided by your ELISA kit into the sample wells. This little piece of equipment makes a big difference in speeding up the whole process.



This pipette allows you to pipette simultaneously into eight wells, saving a lot of time during the ELISA process. Photo by Arista Botha


After incubating your samples the wells have to be washed out properly. You can either manually empty each well and then wash it out with wash buffer using an ordinary pipette (repeat times five!), or (the better option) you can use an automatic well washer, such as the one shown below:




The automatic well washer saves you hours of repeatedly pipetting wash buffer into each well. Photo by Arista Botha.


After washing your samples, you add a substrate solution (consisting of colour reagents), which makes your samples turn blue, if for example, IL-1β is present in your sample. After leaving it to incubate for a set time period you add the stop solution, which changes the colour from blue to yellow.



Adding the stop solution changes the sample colour from blue to yellow. Arista Botha


The final step in the ELISA process is, of course, getting the results! The concept is that the darker yellow your samples, the higher the concentration of the substance you want to measure in your sample (in this case, IL-1β). You put your 96-well plate into a microplate reader, which is set at a certain wavelength, and which will give you optical density (OD) readings. Further calculation of these OD readings ultimately will give you your sample concentration.



The ELISA microplate reader measures the optical density of each well, which you then use to calculate the concentration of your sample. Photo by Arista Botha


After spending the day with the Fever Lab, I feel a lot more confident (and excited) about doing my own ELISAs. I am so glad that we have these equipment and skilled people in our research group and that we can learn from one another. This is why the BFRG is a team and why we do research TOGETHER!