ELISA. MODULE 4 Objective 4.2 Lesson B

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1 MODULE 4 Objective 4.2 Lesson B ELISA Course Advanced Biotechnology Unit Biotechnology Basics Essential Question How is biotechnolog y being used to advance medical diagnostics? TEKS A-K, 2H, 9A, 9C, 10A, 10B Prior Student Learning Immune system, cell Estimated Time 3 hours Rationale ELISA (Enzyme linked immunosorbent assay) is one of many medical diagnostics that doctors and patients use for quick detection of diseases and hormones. The detection of viruses, such as HIV, can now be done in a few hours instead of a few weeks. Objectives Students will: Identify the components and purpose of the ELISA assay. Explain how the body s immune system is involved in riding the body of foreign substances, Virtually perform an ELISA to detect Lupus in three patients. Be able to troubleshoot an ELISA assays, interpret results and explain unexpected results. Engage Key Points: Simulate the spread of HIV and antibody detection from The Biology Project: html Immunology, HIV and ELISA at The Biology Project: Activity 1. Students complete ELISA Pre-lab as an individual assignment. 2. Discuss pre-lab answers to ensure students understand how the immune system works and its relevance in using an ELISA test. 3. Students complete ELISA Virtual Lab. 4. Students complete ELISA Post-lab. 5. Assess students individually using TEST: ELISA Predications. Use food coloring and old wells to create simulated data for a lab practical portion of the test.

2 Optional Wet Lab: Contact your district science vendor to purchase ELISA wet lab kits. These kits are cheaper than other biotechnology kits and offer a skill-based grade based on students results. Results are very sensitive and directly related to student technique. If you order extra kits, allow students to re-perform test if results are wrong the first time. This is a great lab that focuses on following instructions and perfecting lab technique. Assessment ELISA Pre-lab Questions ELISA Virtual Lab Results ELISA Post-lab Questions Correct ELISA wet lab results TEST: ELISA Predictions Materials ELISA: Pre-lab Handout ELISA: Virtual Lab Handout ELISA: Post-lab Handout TEST: ELISA Predictions Accommodations for Learning Differences Visit the Special Populations section of the CTE Career and Technical Education Website: National and State Education Standards*** Science Standards Texas College and Career Readiness Standards Science I. A2, A4, D1, D2, D3, E2 III. B3, D1, D2 IV. E1

3 ELISA Pre-lab Questions Name: 1. Using a website evaluation form, find 3 valid websites (be sure to list your websites in 1b.) that will help you complete the following task: a. Explain why a vaccine exists for the flu virus but not for the HIV virus. Be sure to include how the body s immune system is involved in this process. You may use words, diagrams or concept maps to complete the task. b. Validated Websites: a. b. c.

4 2. Using a website evaluation form, find 3 valid websites (that will help you answer the following questions: a. What does the term ELISA stand for? b. Explain how antibodies, antigens, substrate and serum are used in an ELISA test. c. Briefly outline 10 or fewer steps in performing an ELISA. d. What are ELISAs used for? e. Why are ELISAs preferable to other assays? f. Many companies make ELISA kits to test for particular things. Find an example of an ELISA testing kit. What does it test for and of what value are the test results?

5 3. View the ELISA simulations at: Using the simulations, answer the questions below: a. List the 4 components used to carry out ELISA. b. View a positive and negative result. Explain or diagram the steps involved. c. Is it possible for a person not infected by HIV still have a positive test result? Explain. Positive Control d. Interpret the test result below Negative Control Patient A Patient B Patient C Assay Control O

6 ELISA Virtual Lab Name: Perform the ELISA Virtual Lab found at: 1. What variables can affect the outcome of an ELISA test? 2. Diagram how serum, antibodies, substrate and serum will be used in the ELISA test. 3. What disease will you be testing for in this lab? Explain how the disease affects a person s immune system. 4. What has the ELISA plate been pretreated with? Why?

7 5. Why is it important to have a positive and a negative control? What component is added to each control to identify them as positive or negative? 6. In step 5 why do you incubate the plate at 37 degrees C for 15 minutes? 7. After the first rinse, what remains in the control wells? 8. What problem could arise if the timer is set for less than 15 minutes? 9. What problems could arise if the temperature is too high or too low? 10. How many times is the wash in step 6 completed in a real laboratory? Why?

8 11. What is the identity of the second antibody in step 7? 12. What is the purpose of the substrate-containing enzyme added in step 10? 13. Place an X in each box below to indicate a positive result: Patient A Patient B Patient C Positive Negative 1:2 1:10 1:100

9 ELISA Post-Lab Questions Name: 1. Draw a diagram of the substances that are left in the wells of the positive and negative control. 1. Write a response to each of your patients explaining their test results: 2. Suppose you compare your results with another group that tested fluids from the same potential carriers. What would it mean if the two groups have different results? 3. What was the purpose of washing the plates between additions of each reagent? 4. Explain what is meant by a false positive test. Name one error that would result in a false positive test.

10 5. Explain what is meant by a false negative test. Name one error that would result in a false negative test. 6. Why did you assay your samples in triplicate? 7. What antibody-based tests can you buy at your local pharmacy? 8. Explain how the test below is related to ELISA. 9. Use a separate sheet of paper to explain the difference between indirect and sand which ELISA. Give an example of each. You may use pictures, words or a table.

11 TEST: ELISA Name: What would happen if Predict ELISA results? 1. You forgot to add the secondary antibody. 2. You incubated the ELISA plates at 10 degrees Celsius. 3. Your substrate is left out at room temperature. 4. You only washed your plates once instead of three times. 5. You forgot to wash your plates between adding the primary and secondary antibody. 6. Your patient believes he was infected 2 weeks earlier. 7. Your patient shows positive ELISA results without symptoms. 8. You did not incubate the plate after adding the antigen. 9. Your positive control was negative. 10. Every well on the ELISA plate was positive.