Ex. 28: HIV ELISA, AIDS Diagnostic Tool
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Transcript of Ex. 28: HIV ELISA, AIDS Diagnostic Tool
Ex. 28: HIV ELISA,AIDS Diagnostic Tool
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
ELISA Enzyme-Linked ImmunosorbantAssay
Light chain
Heavy chain
Disulfide bonds
Antigens
Antibody Structure Review
ELISA tests are based on antibody molecules.
HIV ELISA is an Indirect ELISA
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
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
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
• 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
Step TwoAdd controls and patient samples
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 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 constant region of 1° ab • In which wells do you
predict this is happening?
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
ELISA ANIMATION
And . . . .one more ELISA animation
Add purified ag to all the wells. Incubate for 5 min. Rinse
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.
ELISA Procedures Summary
Reagents Summary1. 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
ClearDeterminationOf PositiveAnd NegativeResults