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

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

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

4. Micropipets  Set micropipets to 100 ul and then use micropipets to add and remove samples.

5. Transfer Pipets for rinsing steps  Label transfer pipets PBS 1 PBS 2 PBS 3 PBS 4

6. Let’s ELISA

7. A.  Add.1 ml of antigen to each well  Remove antigen solution  Rinse well one time with PBS Incubate 5 minutes at room temperature

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

9. C.  Remove solutions from each well  Rinse with PBS

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

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

12. OVERVIEW

13. Microplate Readers

14. Examples of Antibody dilutions