Hello! And welcome to part 4 of our common lab techniques sidebar series. By now you’ve hopefully become an expert on virus titers, PCR, and now, to add another acronym to your vocabulary, ELISA. ELISA stands for Enzyme-Linked Immunosorbent Assay. Essentially, it’s a technique to detect if you have a specific protein present in a liquid sample; but before we get started, let’s quickly review two important terms: antigen and antibody.
- Antigen: this is something found on the enemy, be it a virus or bacteria. Because it’s foreign to our bodies, our immune system can detect and organize an immune response against it. Now, an antigen is not necessarily the whole enemy but a part of the enemy, we call this the “epitope” [an antigen is an epitope]. Imagine your immune cells minding their own business, doing their daily patrol, going about your body, then BOOM they see a shoe they’ve never seen before. Strange… they investigate and this is how they realize you have an enemy present in your body.
- Antibody: This is one of the tactics used by your body to fight the enemy once it has detected it. By now you’re an expert on the difference between B and T cells. B cells make antibodies to tack on to the antigen and neutralize it. By neutralize, think of antibodies like dogs who’ve been trained to recognize a specific antigen. Once they see it, they’ll cling to the enemy so that it can’t do any more damage while also signaling to other immune response fighters “Hey! I found him! He’s over here!”.
Now how are these concepts useful to understanding ELISAs? Well, ELISAs use commercially produced antibodies to detect antigens in a sample. These antibodies are specifically produced so that they can detect antigens on one end and are linked to an enzyme on the other. These enzymes are critical because when you add a ‘substrate’ (specific reagent) to them, they produce a colour change so that you can detect if your antigen is present or not. This might sound a bit confusing. But for now but it’ll make sense once you see the steps so stay with me here.
There are 3 main types of ELISAs but all 3 of them use the same concept outlined above.
- Direct ELISA
Direct ELISAs are the simplest of them all: 1 antigen, 1 antibody, 1 substrate. First, you take a liquid sample that you want to test to see if it has the antigen present in it or not. You pour this into your testing well, wait for the proteins to adhere to the bottom, then remove the liquid.
Next, add your antibodies and give it time to bind your antigens. Then, wash away any extra antibodies floating around so that you only have those that are bound to antigens left in your well.
Finally, add your substrate that will turn go from clear to a colour if and when it comes in contact with the enzyme.
A key point here, this entire process will ONLY happen if you have antigen present in your original liquid sample. If there was no antigen, the antibodies wouldn’t be able to bind anything, and hence your substrate would remain clear since no enzyme to turn it blue! The amount of colour change = amount of enzymes = amount of antibodies = amount of antigen (ie. amount of colour change = amount of antigen). Direct ELISAs are quick but there’s a limited number of antibodies with enzymes linked to them available to buy. Since you need a very specific antibody to recognize a specific antigen, this would mean that we’d need millions of antibodies to detect the millions of antigens that scientists deal with! This is where the second type of ELISA comes in handy.
- Indirect ELISA
Indirect ELISAs are very similar to direct ELISAs. The difference is you use 2 antibodies instead of one: a primary antibody and a secondary antibody. Your primary antibody is engineered to detect your antigen with no enzyme and the secondary detects the primary antibody. The secondary antibody also has the enzyme linked to it. Therefore, you have simply added one extra step in the middle.
The primary antibody has no enzyme but is still specific to the antigen. Be sure to wash away any antibodies that are not bound to the antigen before moving on to the next step!
The secondary antibody is specific to the primary antibody and has the enzyme linked to it. Once again, wash away any antibodies that are not bound to a primary antibody!
Finally, business as usual.
Indirect ELISAs are useful since making a primary antibody that only detects an antigen is fairly simple. Since antibody constant and variable regions can be mix and matched, as Deanna explains in this post, the constant region can be switched to be specific for widely available secondary antibodies that are enzyme-linked.
Indirect ELISAs have been especially useful during these pandemic times to identify antibodies that detect SARS-CoV-19 antigens- i.e., researchers may know the antigen SARS-CoV-19 but not which antibody binds to it. All human antibodies have the same constant region, read more about it here. So the antigen will bind the antibody you’re looking for and the secondary antibody will bind it as well. Think of it as a puzzle, the antigen, primary antibody and secondary antibody are all pieces that interlock, one after the other. What if you knew the first puzzle piece (SARS-CoV-19 antigen) and the last piece (an antibody that can bind all human antibodies), and you have a whole box of possible puzzle pieces (the serum sample of a patient that has tons of antibodies). You’d be able to find out which puzzle fits in between! This is important for vaccine and drug research!
- Sandwich ELISA
Sandwich ELISAs are called this because well, you create a sandwich, I know, very creative naming here in the scientific community. In this ELISA, rather than adhering the antigen to the bottom of the well, you start by adhering an antibody (we call this one the capture antibody), then add antigen and continue as either a direct or indirect ELISA.
Now add your antigens! Be sure to wash away any remaining antigens that are not bound to your antibody before moving on to the next step!
Now carry on as either a direct or indirect ELISA on top!
Sandwich ELISAs are useful if you want to be really really specific about the antigen you’re detecting since it requires 2 antibodies (capture and primary) to detect it!
Overview of ELISA. (n.d.). Retrieved May 19, 2020, from https://www.thermofisher.com/ca/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html
ELISA for Novel Coronavirus (2019-nCoV, SARS-Cov-2) Causing an Outbreak of Pneumonia (COVID-19). (n.d.). Retrieved May 21, 2020, from http://www.epitopediagnostics.com/covid-19-elisa