BIOTECH Project, University of Arizona
ELISA Assay
Name:
Period:
Using the ELISA Assay for Disease Detection
In this laboratory you will use the ELISA (Enzyme Linked ImmunoSorbent Assay) for detecting the presence of an antigen, such as a disease-related agent, from a sample of body fluid. You will be given a solution that represents your body fluid. You will exchange body fluid with three other individuals. The ELISA test will be used to detect a simulated disease agent in the mixed body fluids. Finally, you will analyze the class data set to determine identity of the original carrier(s).
This assay is similar to that used for the AIDS test, in which an ELISA is used to detect individuals infected with the virus that causes AIDS (HIV).
Materials:
Each student should have the following items before starting the laboratory:
*Microcentrifuge tube containing "body fluid" (BF)
*Disposable transfer pipet
Each group of three students should also have:
*ELISA test plate
The class should have
*Dropper bottles with positive control solution (+ )
*Dropper bottles with negative control solution ( -)
*Paper towels
*Dropper bottles with Antibody solution (Ab)
*Washing solution (in wash bottle)
*Dropper bottles with Color reagent solution (TMB)
Procedure:
Exchanging Sharing Solutions
1. At the start, you'll have a transfer pipet and a tube containing a solution that represents your "body fluids."
Be sure to choose students from all over the class, and not just in your immediate area. Wait for the instructor's signal before exchanging fluids.
2. Find one other student in your class and exchange your solution from your 'body fluid' tube with him or her. Use transfer pipet to swap half of your 'body fluid'.
#1 sharing:
3. At the instructor's signal find a different student to exchange your sharing solution with.
#2 sharing:
4. At the instructor's signal exchange your sharing solution with one more student.
#3 sharing:
ELISA Test
After all three contacts have been completed, you will do an ELISA test on your sharing tube fluid.
1. Form a group (usually three people) at your lab station. Your group will run an ELISA assay on your mixtures of body fluids from each student in your group using an ELISA plate.
Samples Loaded Results
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2. Using your transfer pipet, add three drops of your sharing tube fluid into each of three wells. Be sure to record which wells contain your fluid to avoid confusing your wells with another person's.
3. Each group will also do one positive and one negative control. Using the dropper pipets for each control, add twodrops of a positive test solution (+) into three wells and two drops of a negative test solution (-) into a different set of three wells.
4. After everyone has added their solution, leave the plate on the lab table undisturbed for five minutes.
5. Shake off the fluid into a nearby sink or designated container, making sure that the fluid has emptied from each well. Tap the plate upside down onto the paper towel to remove any excess liquid or bubbles.
6. Wash your group's plate 3 times and shake off.
7. Using the dropper pipet for the antibody solution add two drops of the antibody solution (Ab) to each well.
8. Allow five minutes incubation time on the lab table and then shake off the fluid.
9. Wash your group's plate 3 times and shake off.
10. Using the dropper pipet for the TMB solution add two drops of the color reagent solution (TMB) to each well.
11. Watch your plate for the next 5 minutes. What happens? Record your results in the table on page 2.
Laboratory Questions
Each group will record the results of the tests. Be sure to record whether you are infected. You will also record your test results and list of partners on the blackboard.
1. Given the classroom data, who are the original infected carriers?
2. Why were positive and negative controls needed?
3. What was the purpose of washing the plates between addition of each reagent?
4. Explain what is meant by a false positive test. Name one error that would result in a false positive test.
5. Explain what is meant by a false negative test. Name one error that would result in a false negative test.
6. How can you protect yourself from transmitted diseases?
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