Adapted from Source: Cynthia Harley, Ph.D. and Anthony Auletta, M.S.
ntroduction to ELISAs: What, How, and Why
An ELISA (short for Enzyme-Linked Immunosorbent Assay) is a common and powerful diagnostic technique that allows scientists to test whether an unknown sample contains a specific antigen. The antigen is often a protein that is found on the surface of a pathogen, like a virus or a bacterium. Thus, by testing for the presence of these antigens, we can determine whether our samples have encountered the pathogen. This means that an ELISA can be used to diagnose someone with a disease.
So, how does an ELISA work? There are several steps, but the underlying principle is simple—by the end of the procedure, samples that contain the antigen will change color, while samples that do not contain the antigen will not change color.
Here is how it’s done:
The antigen in your fluid (this is on the surface of infected cells) will be bound to an antibody (the dye that I am adding). When the two bind, a reaction happens, making it change color. This color change does not happen if the antigen isn’t there (for instance, if the person does not have the disease).
Required Materials
Test tubes
Pipettes
Well plates
NaHCO3 (baking soda) solution
Cabbage dye solution (pH indicator)
Colored tape (3 colors)
Sodium citrate buffer
The Situation
You have reason to suspect that someone in your class has recently become infected with the highly contagious disease, Harley-Auletta Virus! According to Dr. John A. Contagion, Harley-Auletta Virus can result in fever, spasms, madness, coma, and even death—but the disease can stay dormant for many years, so it’s possible to be infected without even knowing it.
Since the symptoms can be delayed for so long, Dr. John A. Contagion decides to use a series of ELISAs to determine which class members have contracted Harley-Auletta Virus and how quickly the disease is spreading throughout the population.
Harley-Auletta Virus can be spread from person to person through respiratory droplets, which means it spreads through coughing, singing, and even talking! At your desk, you will find a series of tubes, each of which contains a colorless liquid. These tubes represent your respiratory droplets. In a few moments, you will exchange droplets with other members of the class through simulated contact. Most of you have pure water in your tubes, which signifies that you are not infected (yet). However, one mysterious student—Patient Zero—has a bit of baking soda dissolved in their tubes. For the purposes of our simulations, this means that this person is infected with Harley-Auletta Virus.
As you perform the simulations that follow and run your mock ELISAs, think about how the different conditions of each simulation may affect how quickly the disease spreads from Patient Zero to other people in the class. At the end of each simulation, we will collect some data on the infection spread, and use the data to create a series of graphs.
NOTE: Each of your tubes should be marked with a number; this is your “patient ID,” and you will need it when you put your fluid samples in the wells on the well plates. Also, note that we will be using different tubes and pipettes for each of the three simulations: ORANGE tape denotes materials for Simulation 1, BLUE tape is for Simulation 2, and YELLOW tape is for Simulation 3.
Simulation 1 Procedure
For the first simulation, we will explore how the disease spreads when the level of mingling is low—one contact per round.
For this simulation, only use the tubes and pipettes that have the ORANGE tape on them. The other tubes and pipettes will be used in later simulations, so set them aside.
Pair up with one other person in the class. Use your orange pipette to collect some of the liquid from your orange tube and squirt it into your partner’s orange tube. Allow your partner to squirt some of his or her liquid into your tube as well. This is how we will simulate close contact.
Use your pipette to place a small sample (one drop will do) from your tube into Row A of the provided well plate marked “Simulation 1,” in the column that corresponds to the number on your tube. For example, if you are using Tube #9, place your sample into well 9A.
Using the same tube and pipette, repeat the previous steps with another student in the class. When you are done, place a sample from your tube into Row C of the well plate, right under your first sample.
Repeat this process one more time, so you’ve has contact with a total of three different people in the class and you have three samples in your column of the well plate (Rows A, C, E—leaving one row between samples to avoid contamination).
Once everyone has all their samples in the wells, the instructor will administer a drop of cabbage dye into each well. Cabbage dye solution is normally bluish-purple, but it turns green in the presence of baking soda. Thus, if the sample in a well turns green, it means that it has baking soda (virus) in it. If the sample in a well stays bluish-purple, then there is no baking soda (virus) in it.
Record the number of infected individuals (= green wells) for each time point in Table 1.
Question
Based on the data, what happened?
Simulation 2 Procedure
For this simulation, we will do the same thing that we did for Simulation 1, with one big difference: now, everyone in the population is in contact with multiple partners every day!
Prediction: What do you think this means for the spread of disease?
For this simulation, only use the tubes and pipettes that have the BLUE tape on them.
Pair up with one other person in the class and simulate contact with him or her just like you did in Simulation 1 (make sure to use the blue pipettes and tubes this time). Then, immediately pair up and make contact with two additional classmates.
After your third contact, use your pipette to place a small sample (one drop will do) from your tube into Row A of the provided well plate marked “Simulation 2,” in the column that corresponds to the number on your tube. For example, if you are using Tube #9, place your sample into well 9A.
Repeat this process two more times, making sure that you make contact with three different people during each round. By the end, you should have made contact with a total of nine different people in the class, and you should have three samples in your column of the well plate (Rows A, C, E—leaving one row between samples to avoid contamination).
As in the previous simulation, the instructor will place a drop of cabbage dye into each well of the plate.
Record the number of infected individuals (= green wells) for each time point in Table 2.
Question
Based on the data, what happened?
Simulation 3 Procedure
Now, we will examine the effects of using a vaccine on the spread of the disease. You will proceed with the same scenario as Simulation 2 (three contacts each round, nine total), but some of you will receive a vaccination (sodium citrate buffer). We will complete three different simulations, and in each one, the number of people who receive a vaccination will be different.
For these simulations, only use the tubes and pipettes that have the YELLOW tape on them.
Prediction: What effect do you think vaccines will have? Does it matter how many people get them?
Simulation 3A—if your tube number is 3, 6, 9, or 12, you get the vaccine
Simulation 3B—if your tube number is 15 or less, you get the vaccine
Question
Based on the data, what happened?
Discussion Questions
What component of an actual ELISA did the starch represent in our experiment? What did the cabbage dye represent?
How did the spread of disease differ between Simulations 1 and 2? What is the reason for this difference?
How did vaccination influence the spread of disease?
Did it matter how many people were vaccinated? Why?
What would happen to the spread of disease if some individuals had previously been exposed to the virus strain? Explain your reasoning.