Enzyme Linked Immunosorbent Assay

1. Enzyme Linked Immunosorbent Assay ELISA Detection of Anti-HIV (This outline summarizes key points of ELISA protocol as outlined in the handout.)

2. ANTI-HIV Simulate LABORATORY • Introduction to the protocol • Laboratory Steps

3. Enzyme Linked Immunosorbent Assay (ELISA) • A diagnostic test for Antibodies to HIV Antibodies

4. Antibodies Antigens Immune system Non-self

7. Objective: to use the ELISA to detect anti-HIV from 2 donors.

8. Supplies needed /group • 4 transfer pipets • 1 transfer • 1 set of samples • 1 microtiter plate (12 wells) • 1 beaker of Phospate buffered saline (PBS) • 1 empty beaker for “waste”

9. Transfer pipets • Try to approximately add the • Same volume to each well. • Approximate volume • 0.l ml

10. Transfer Pipets for rinsing steps • Label transfer pipets • PBS 1 • PBS 2 • PBS 3 • PBS 4

11. Let’s ELISA

12. A. • Add .1 ml of antigen to each well • Remove antigen solution • Rinse well one time with PBS Incubate 5-10 minutes at room temperature

13. PBS + control Donor 1 Donor 2 Row 1 Row 2 Row 3 Row 4 B. Add 0.1 ml of Incubate 10-15 min. at 37C Row 1 Row 2 Row 3 Row 4 NOT IN A WATER BATH!!

14. C. • Remove solutions from each well • Rinse with PBS

15. D. • Add .1 ml of labeled 2nd Ab to each well • Remove 2nd antibody solution • Rinse wells with one time PBS Incubate5-10 minutes at 37C

16. E. • Add .1 ml of substrate to each well • Identify wells with anti-HIV 5-10 minutes at 37C

17. OVERVIEW

18. Microplate Readers

19. Examples of Antibody dilutions

20. Hand-In: • A drawing of the steps involved in the ELISA • The results of your laboratory results • The interpretation of your results • Provide an example of how you could obtain a false positive and a false negative result.