Immunology and the Spread of Diseases

Objective:  This lab will allow you to understand better how diseases are spread from person to person and how to use the ELISA assay to test whether a person is infected with a specific pathogen such as the HIV virus.  No actual pathogens are used in this lab.

Procedure Overview:  Each person will be given a clear tube with fluid in it.  These tubes represent each person’s body fluids.  One of the tubes will have an antigen (small protein) from a simulated pathogen. 

1. In part 1, you will then share your “body fluids” with 3 other people in 3 steps.  Thus, the “infected” person will be infecting other people, who then go on to infect, other people. 
2. In part 2, you will conduct the ELISA assay on your “body fluids” to see if you are infected. 
3. In part 3, you will try to determine who started the “epidemic.”  By making a table of who shared body fluids with whom and who is infected at the end of this process, you determine who received the tube with the antigen in it.  This procedure will narrow the possibilities to 2 students who could have brought the “infection” into the classroom.
4. In part 4, a medical history will determine which of the 2 students first had the “infection.”

PART 1 – Sharing of “Body Fluids”

1. Find a partner with whom to share your sample. 
2. Upon the signal from your lab instructor, share your sample with your partner by using a transfer pipette to combine the two samples in one student’s tube and then transfer half of the sample back to the other student’s tube.  Throw the transfer pipette away in a trash can. 
3. In Table 1 below, write the name of the person with whom you shared “body fluids” and their tube number.
4. Find a second partner in another part of the room with whom to share your sample.
5. Upon a signal from your lab instructor, share your sample following the procedure explained in step 2, above.  Throw the transfer pipette away in a trash can. 
6. Write the name of the second person with whom you shared “body fluids” and their tube number in Table 1 as before.
7. Find a third partner with whom to share your sample
8. Upon a signal from your lab instructor, share your sample following the procedure explained in step 2, above.  Throw the transfer pipette away in a trash can.  
9. In table 1 below, write the name of the third person with whom you shared “body fluids” and their tube number as before.

Table 1. Students you shared fluids with

STEP 2 - ELISA Assay
In this part of the lab exercise, you will run an ELISA test on your shared samples that you have in your tube.  Each sample will be assayed in triplicate, or three times.  You will have one assay plate for every 4-6 students.  An assay plate has 8 columns with three wells each, and thus has columns for 2 controls and up to 6 students.  

Table 2.  ELISA Assay Form

Procedure

Note: Be sure you record the placement of each control and sample!

1. Using your blue pipettor and a fresh pipette tip, place 50 µL (microliters) of the  positive control (green tube) into the each of the 3 wells in the first column on your assay plate.  Discard the pipette tip.  Record the location on your assay form (Table 2).
2. With a fresh pipette tip on your blue pipettor, place 50 µL of the negative control (pink tube) into each of the 3 wells in the second column on your assay plate.  Discard the pipette tip.  Record the location of the controls on the ELISA assay form (Table 2). 
3. Using the 50-ul fixed volume pipette with a fresh pipette tip for each person, everyone in your group should add 50 µL of the shared fluid from their tubes into each of 3 wells in one column.  Record the location of each person’s column on the ELISA assay form (Table 2) below. 

Make sure each person uses a fresh pipette tip.

4. Let the plate sit undisturbed on the lab table for 15 minutes. 
5. During this time, go to the computer up front, and enter your name next to your tube number on Table 3, then enter the tube numbers of the students with whom you shared “body fluids.”
6. After 15 minutes, shake off the fluid into the sink at the side of the room.  To empty the wells completely, flick the plate (well side down) against the edge of the sink. (You can be pretty rough with these plates.)
7. Use the squeeze bottle of wash solution to wash each of the wells three times, shaking and flicking out the wash buffer between each wash. 
8. After washing the wells, tap the plate firmly upside down on a stack of paper towels to remove the remaining fluid.  When you finish the drying procedure, there should be no more liquid appearing on the paper towel when you tap the plate.
9. Using a new pipette tip, add 50 ul of antibody solution (orange tube) to each well.  Let the plate sit on the bench for 5 minutes.
10. Shake off the fluid and wash the plate as in steps F-H above.
11. Using a new pipette tip, add 50 ul of color reagent solution (purple tube) to each well.  Let the plate sit on the bench for 10 minutes.
12. Observe the results.  Negative wells (“uninfected” and the negative controls) will remain totally clear.  Positive wells (“infected” and positive controls) will turn blue.
13. Go to Table 3 on the computer and where you wrote your name and the tube numbers of the 3 people you shared with and put a – next to your name and tube number if the results are negative and a + next to your name and tube number if the results are positive.
    

Table 3: ELISA Assay Results