Ex. 28: HIV ELISA, AIDS Diagnostic Tool

1. Ex. 28: HIV ELISA, AIDS Diagnostic Tool

2. Human Immuno-deficiency Virus (HIV) • First diagnosed in 1981 • Over 20 million deaths worldwide, over a half million in the United States • Over 40 million currently infected, over a million in the United States • Education effective in limiting the spread of HIV/AIDS

3. Heavy chain Disulfide bonds Light chain ELISAEnzyme-LinkedImmunosorbantAssay Antibody Structure Review ELISA tests are based on antibody molecules. Antigens

4. HIV ELISA is an Indirect ELISA

5. ELISA-HIV TestDetecting Antibodies in Serum • After 4-8 weeks of exposure to the HIV virus: body will have produced detectable level of antibodies against HIV • HIV-ELISA detects presence of serum antibodies against HIV protein antigens

6. Step OneLabel wellsand add antigen Label the 12-well strip: • First 3 wells: positive controls “+” • Next 3 wells: negative controls “-” • Remaining wells patient samples (3 wells for each patient) Transfer 50µl of purified antigen (AG) into all 12 wells Wait 5 minutes for the antigen to bind

7. Microplate Strips • Microplate strips are made of polystyrene • Hydrophobic side chains in amino acids bind to the polystyrene wells

8. WASH • Remove the liquid from the sample wells by tipping the microplate strip upside down and discarding the solution into a beaker. • Firmly tap the strip a few times upside down onto a paper towel. • Discard the paper towel. • Using a disposable transfer pipette almost fill the wells with wash buffer. • Remove the wash buffer following the procedure above. Always discard the used paper towels • Repeat the wash step

9. Step Two Add controls and patient samples • Add 50 µl of positive control to 1st three wells • Add 50 µl of negative control to 2nd three wells • Add 50 µl of patient sample A to 3rd set of three wells • Add 50 µl of patient sample B to last 3 wells • Incubate at room temperature for 5 minutes. • Wash twice

10. Wash Buffer • Wash buffer contains phosphate buffered saline (PBS) to keep ABs in stable environment that helps keep their structure • Also contains Tween 20: a nonionic detergent that helps to remove non-specifically bound proteins (reduces background)

11. Step ThreeAdd enzyme-linked AB • Add 50 µl of the enzyme-linked secondary antibody to each well • Incubate at RT for 5 minutes. • 2° ab(enzyme-linked antibody) will only bind to primary ab(serum antibody) • 2° ab specifically recognizes constantregion of 1° ab • In which wells do you predict this is happening?

12. Step FourAdd enzyme substrate • Wash the enzyme-linked secondary antibody from polystyrene wells as before • WASH 3X • Add 50µl of the enzyme substrate to each well • Incubate at room temperature for 5 minutes • Positive samples will begin to turn blue

13. ELISA ANIMATION And . . . .one more ELISA animation

14. Add purified ag to all the wells. Incubate for 5 min. Rinse ELISAProcedures Summary Add serum antibodies (patient samples) to the appropriate wells. Incubate for 5 min. Rinse Add the enzyme-linked antibody to all wells. Incubate for 5 min. Rinse Add enzyme substrate to all wells. Incubate for 5 min.

15. Purified HIV Antigen • Primary antibody (Patient serum samples) • Secondary antibody: conjugated polyclonal anti-human antibodies made by goats. Conjugate is horseradish peroxidase (HRP) • Substrate for HRP: 3,3’,5,5’ – tetramethylbenzidine(TMB) – a colorless solution that turns blue when oxidized by HRP Reagents Summary

16. ELISA KitResults Clear Determination Of Positive And Negative Results The End