Virtual Immunology Lab
http://www.hhmi.org/biointeractive/vlabs/immunology/index.html
Follow the directions in the video to the left. Answer the following
questions using the references tabs as shown below.
You also may want to open up
http://www.biology.arizona.edu/immunology/activities/elisa/technique.html to help you
visualize what’s going on at a molecular level while you go through the lab procedure.
1. The disease you will be testing for is systemic lupus erythematosus. What are some of the
symptoms of this disease? What’s going wrong in the body?
2. Lupus patients’ immune systems produce antibodies that bind to molecules in their own cell
nuclei. (These molecules are known as nuclear antigens, and they include things like DNA
and histone proteins that wrap around the DNA.) Why is this a bad thing?
3. Why is centrifuging the blood samples necessary? (In your own words, please! Don’t just
copy it off the webpage!)
4. What does serum consist of? What major part of the blood is NOT in the serum?
5. What are you diluting the serum with in Step 2?
6. Why did we make several different dilutions (one very diluted, one kinda diluted, and one
barely diluted at all) for each patient’s serum? (Hint: you may want to come back to this
question after you’ve finished the whole simulation.)
7. In step 3, the ELISA plate already has lots of copies of the nuclear antigens bound to the
bottom of each well. Why is it necessary to use this kind of plate instead of a completely
empty one?
8. A “positive control” is a sample that will give you a positive result at the end of the test
(assuming that you did all of the steps correctly). What did you put in the positive control
well that is guaranteed to give you a positive result?
9. A “negative control” is a sample that will give you a negative result at the end of the test
(assuming that you did all of the steps correctly). What did you put in the negative control
well that is guaranteed to give you a negative result?
10. Draw the molecules that should be present in the positive and negative control wells at
this point. (Hint: the wells in this ELISA plate come pre-coated with nuclear antigens.)
11. Why is the ELISA plate incubated at 37° C? Why not room temperature?
12. Why would we want to wash out any antibodies that haven’t found an antigen to bind to?
What would happen if we didn’t wash them out?
13. In step 7, we need an antibody that will bind to the stalk
of the patient’s antinuclear antigen antibodies (if they in fact have the disease). Why did we use antibodies that
a rabbit made to recognize these human antibodies? Why don’t we use human antibodies
instead? (Hint: all human antibodies have the same stalk, no matter what antigen their top
part is supposed to bind to.)
14. When the plate is incubated a second time, why is the time so important? What would
happen if it was heated less than 15 min? more than 15 min?
15. Draw the molecules that should be present in the positive and negative control wells at this
point.
16. What happens when HRP-substrate and the horseradish peroxidase enzyme that’s attached to
the rabbit antibody come in contact with each other?
17. Draw the molecules that should be present in the positive and negative control wells at
this point
18. Check the boxes that are colored in the ELISA plate:
19. Summarize your results. Which patients have the disease for sure? Which do not? Which
patient(s) are you not sure about and why?