• 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
ELISA
Antibody Structure Review
Enzyme-Linked Immunosorbant Assay ELISA tests are based on antibody molecules.
Heavy chain Disulfide bonds
Light chain
Antigens
ELISA-HIV Test Detecting 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
Step One
Label wells and 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
Microplate Strips
• Microplate strips are made of polystyrene • Hydrophobic side chains in amino acids bind to the polystyrene wells
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
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
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)
Step Three Add enzymelinked AB
• Add 50 µl of the enzyme-linked secondary antibody to each well
• Incubate at room temperature for 5 minutes. • Secondary AB (enzyme-linked antibody) will only bind to primary AB (serum antibody) • Secondary AB specifically recognizes the constant region of the primary AB • In which wells do you predict this is happening?
Step Four Add 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
ELISA ANIMATION
And . . . . one more ELISA animation
Add purified ag to all the wells. Incubate for 5 min. Rinse
ELISA Procedures 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.
Reagents Summary 1. Purified HIV Antigen 2. Primary antibody (Patient serum samples) 3. Secondary antibody: conjugated polyclonal anti-human antibodies made by goats. Conjugate is horseradish peroxidase (HRP) 4. Substrate for HRP: 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless solution that turns blue when oxidized by HRP
ELISA Kit Results Clear Determination Of Positive And Negative Results
Materials needed per table (serves 4 students) Items
