Ex. 28: HIV ELISA, AIDS Diagnostic Tool
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1 Ex. 28: HIV ELISA, AIDS Diagnostic Tool
2 Human Immunodeficiency 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
4 ELISA Enzyme-Linked Immunosorbant Assay Antibody Structure Review Heavy chain ELISA tests are based on antibody molecules. Disulfide bonds Light chain Antigens
5 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
6 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
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 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
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 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?
12 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
13 ELISA ANIMATION And.... one more ELISA animation
14 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.
15 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
16 ELISA Kit Results Clear Determination Of Positive And Negative Results
17 Materials needed per table (serves 4 students) Items Contents Number Eppendorf tubes (labeled with Patient serum samples to be tested patient s initials) for antibodies (0.25 ml) 2 Eppendorf tube (labeled AG) Purified antigen (1.5 ml) 1 Eppendorf tube (labeled +) Positive control (0.5 ml) 1 Eppendorf tube (labeled -) Negative control (0.5 ml) 1 Eppendorf tube (labeled SA) Secondary antibody (1.5 ml) 1 Eppendorf tube (labeled SUB) Substrate (1.5 ml) 1 12-well microplate strip µl adjustable micropipette 2 Appropriate tips for pipette box Disposable plastic transfer pipettes 2 Small empty beaker 1 Phosphate buffered saline (PBS) ml wash buffer in beaker with 0.05% Tween 20 1 Paper towels many Black marking pen 1 Work as a table team
18
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