Ex. 27: 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 E nzyme- L inked I mmuno s orbant A ssay 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 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 / Microtiter Plates are made of polystyrene Hydrophobic side chains in amino acids bind to the polystyrene wells

Most important step: WASH 1.Remove liquid from sample wells by tipping microplate strip upside down and discarding solution into big glass petri dish 2.Firmly tap strip a few times upside down onto a paper towel 3.Discard paper towel 4.Using disposable transfer pipette almost fill wells with wash buffer 5.Remove wash buffer following procedure above. Always discard the used paper towels 6.Repeat wash step

Add 50 µl of positive control to 1 st three wells Add 50 µl of negative control to 2 nd three wells Add 50 µl of patient sample A to 3 rd 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 Two Add controls and patient samples

Wash Buffer contains … Phosphate buffered saline (PBS) to keep abs in stable environment that helps keep their structure Tween 20: Nonionic detergent that helps to remove non- specifically bound proteins (reduces background)

Step Three Add 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 binding will occur?

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. Wash. Add serum antibodies (patient samples) to the appropriate wells. Incubate for 5 min. Wash. Add the enzyme-linked antibody to all wells. Incubate for 5 min. Wash. Add enzyme substrate to all wells. Incubate for 5 min. ELISA Procedures Summary

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