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