Immunology Day Senior Activity – ELISA Assay
Date: April 27, 2012
Introduction:
There has recently been a disease outbreak in Southern Ontario. The symptoms which
have contracted the virus exhibit symptoms such as fever, swelling of the lymph nodes,
weakness, and bumps of the skin. Scientists have identified the virus responsible for the disease
and sequence analysis confirmed that it is a newly discovered strain of herpes virus (like
chickenpox). Scientists working at the Department of Immunology at the University of Toronto
have isolated an antibody which is capable of neutralizing this virus and so clinical trials have
begun to eradicate the pandemic. In order to study the cause of the outbreak and prevent it from
happening, we must find exactly where it came from to study what environment harboured it.
As a research scientist working for Let’s Talk Science, you have been assigned two tasks.
The first is to determine how infectious this pathogen really is. Secondly, you must track the
virus down to the initial individual infected. LTS and the Dept. of Immunology have provided
you with access to patient’s bodily fluid and dates of when they were admitted to the hospitals in
the region. They have also given you access to the antibody which they have generated to fight
the virus. Let’s Talk Science has trained researchers to help you in your quest.
Materials and Methods
Materials:
Bodily fluid from collection of patients (in 1.5 ml microcentrifuge tube)
Positive control (in microcentrifuge tube)
Negative control (in microcentrifuge tube)
Anti-virus antibody solution + substrate reagent
96-well microplate
Pipettes and tips for your experiments
Marking pen
Gloves **Make sure students are wearing them at all times when handling reagents**
Methods:
1. Students will come up to the front of the classroom to do the activity with the LTS
volunteer.
2. LTS Volunteers will ask 6 students to represent the 6 initial patients.
3. Load 20 ul of each of the 6 samples into empty wells on the 96-well microplate.
4. Give empty 1.5 ml microcentrifuge tube to another 6 students and ask them to represent
“naïve” individuals.
5. Ask them to share their samples with each other but remember to ask them to keep track
who they have shared their samples with. You may mix around 100 ul of sample each
time there is sharing.
6. In the end, you should have 12 tubes that have all been shared amongst each other.
7. Load 20 ul of each of the samples in an empty well.
8. Ask your LTS volunteer to develop the assay by adding 150 ul of substrate solution to
each well with mixed sample in it.
9. Can you figure out who the initial person infected was?
10. Add 150 ul of substrate solution with the wells that have original patient samples in
them.
11. Confirm your experiment by developing your positive and negative control.
Results (Example of sample loading):
Questions:
1.
2.
3.
4.
5.
Based on your results, how many individual(s) were infected before the exposure?
Based on your analysis, how many individual(s) were infected after the exposure?
How virulent (ability to infect) would you say the virus is?
How reliable is your analysis? (Hint: think of your controls)
Would you say that the antibody developed by the Scientists is a highly specific one?
How can you test this?
6. Would you consider the ELISA Assay a fast and effective one for observing and tracking
disease outbreaks?