Review Antibody structure and function: Janeway Chapter 3 pp 103-115 and Chapter 4 pp 156-164 or Parham Chapter 2 pp 37-47, 57-61.
Read about ELISA in the ToolBox and perform one of the virtual ELISA labs (links on the ELISA ToolBox page). [Bring a copy of the ELISA page with you - it will be helpful in discussion.]
Questions to answer and turn in:
You have vaccinated several human subjects with HIV gp120, the envelope protein that allows HIV to bind to CD4 (helper) T cells.To test the efficacy of the vaccine you use an ELISA to test for antibodies to gp120.
Red dots = purified HIV gp120; yellow Y's = human antibody molecules in the serum from your test subjects; blue Y's = rabbit-anti human gamma chain; * = enzyme covalently attached to rabbit anti-human gamma chain. Photo of four rows of the 96-well plastic plate in which the ELISA is done shows results for four test subjects, one in each row. Patient serum is diluted 1:10 in the far left wells and diluted two-fold in each well to the right for the first 10 wells: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5280. The 11th well in each row is a positive control and the 12 well in each row is a negative control.
1. How does the HIV gp120 stick to the 96-well plate? What must you do to prevent antibody from sticking in this fashion? What would be your results if antibody did stick to the plastic instead of to the gp120?
2. Do all the antibodies in the patient serum (yellow Y's) bind to the antigen? To what antigens besides HIV gp1120 might the subject have antibodies?
3. What is the isotype of the patient antibody that the enzyme-linked rabbit anti-human gamma chain antibody binds? What other isotypes are present in patient serum? If the enzyme-linked rabbit anti-human gamma chain antibody is also an IgG, why would it not bind to its own isotypic epitope?
4. Where does the color change come from in the microtiter plate wells? What actually changes color?
5. The antibody titer is the highest dilution that gives a positive result. What are the anti-gp120 titers for the four subjects? Why are they all different?
6. What antigen, antibody and second antibody would you put in a positive control (a test that you would expect to be positive)? What antigen, antibody and second antibody would you put in a negative control (a test that you wou8ld expect to be negative)?
7. What control could you do to show that the patient antibody is binding specifically to the antigen and not nonspecifically to the plastic plate? What control could you do to show that the rabbit anti-human gamma chain is binding specifically to the patient Ig and not to the antigen or the plastic plate?