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Laboratory #2: ELISA Immuno Explorer

Wheeler High School The Center for Advanced Studies in Science, Math & Technology. Laboratory #2: ELISA Immuno Explorer. Lab Timeline : Intro to ELISA H1N1 Genetics ELISA Lab Overview ELISA Assay. Post-AP DNA/Genetics – Ms. Kelavkar. Day #1: Introduction to ELISA.

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Laboratory #2: ELISA Immuno Explorer

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  1. Wheeler High SchoolThe Center for Advanced Studies in Science, Math & Technology Laboratory #2: ELISA Immuno Explorer Lab Timeline: Intro to ELISA H1N1 Genetics ELISA Lab Overview ELISA Assay Post-AP DNA/Genetics – Ms. Kelavkar

  2. Day #1: Introduction to ELISA • ELISA stands for enzyme-linked immunosorbent assay • Also used to test for pregnancy, drugs and genetically modified organisms (GMO’s) • Antibody based test to diagnose disease such as HIV, SARS, H1N1, STD’s, anthrax Post-AP DNA/Genetics – Ms. Kelavkar

  3. Introduction to ELISA • In this lab you will share simulated ‘body fluids’ with your classmates to see how easily it is to pass on H1N1. • Then you will perform an ELISA test to determine if you have been exposed to this contagious disease. Post-AP DNA/Genetics – Ms. Kelavkar

  4. ELISA tests use antibodiesto detect the presence of a disease. Molecules that cause your body to start the immune response are called antigens. Heavy chain Disulfide bonds Light chain Introduction to ELISA Antibody Structure Antigens

  5. After becoming infected, within days your body will have produced millions of antibodies. Recall from AP Bio that antibodies are proteins that recognize the antigen and bind to it. Introduction to ELISA

  6. Introduction to ELISA • In this lab we will use antibodies to determine the presence of an antigen (H1N1). Immunologists inject chickens, goats, rabbits or sheep with the antigen and then harvest the antibodies in their blood to use as a diagnostic tool in the lab.

  7. Introduction to ELISA • The antibodies used to recognize antigens are called primary antibodies. • Secondary antibodies, which come from another species, bind to primary antibodies when injected.

  8. Let’s See How ELISA is used in determining Pregnancy… • http://www.sumanasinc.com/webcontent/animations/content/pregtest.html

  9. Day #2: H1N1 Genetics & Fluid Transfer • The ‘swine flu’ is actually a combination of avian (bird) and swine influenza genes.

  10. Why H1N1? • Influenza and their subtypes are named & classified based on their surface proteins. • Both Hemagglutinin and Neuramindaseare surface proteins found on the influenza virus. Influenza Virus

  11. Hemagglutinin – an antigenic glycoprotein Binds the virus to the cell it’s trying to infect Neuramindase – allows the virus to inject it’s viral genome into the host and replicate Surface Proteins

  12. The ‘swine flu’ has never been isolated from pigs!!! The genes of human ‘swine flu’ VERY SIMILAR to swine influenza thus the media dubbing it ‘swine flu’. Therefore, it is unknown if the swine flu is actually zoonotic. One Possibility Did You Know…

  13. Genetic re-assortment occurs when more than 1 virus infects a cell Viral DNA/RNA gets mixed & matched up giving various genetic combinations So if it didn’t come directly from pigs…where the heck did it come from?

  14. ELISA Assay Overview: Step #1 • Obtain a test-sample • Label the 12-well strip: • First 3 wells: positive controls “+” • Next 3 wells: negative controls “-” • Remaining wells to identify test-samples Proteins in the samples will bind to the wells via hydrophobic interaction.

  15. ELISA Assay Overview • Microplate strips are made of polystyrene • Hydrophobic side chains in amino acids bind to the polystyrene wells • No special coating is needed

  16. ELISA: Step #2 • Remove samples from wells by firmly tapping them on a paper towel • Discard the top paper towel • Using a disposable transfer pipette wash wells with wash buffer • Remove wash buffer by firmly tapping the wells on a paper towel • Discard the top paper towel • Repeat wash step

  17. Step #3: Add controls to your samples • Add 50 ul of positive control to 1st 3 wells • Add 50 ul of negative control to 2nd 3 wells • Add 50ul of student sample A which represents students serum sample to 3rd set of 3 wells • Add 50ul of other student sample B which represents that student’s serum sample to last 3 wells • Samples are left in wells for 5 minutes.

  18. Step #4: Wash antibody & add enzyme-linked antibody • Wash the primary antibody from polystyrene wells as before • WASH 2X • Add 50ul of the enzyme-linked secondary antibody to each well • Wait 5 minutes

  19. Step #4: Add enzyme substrate • Wash the enzyme-linked secondary antibody from polystyrene wells as before • Using a disposable transfer pipette wash wells with wash buffer • WASH 3X • Add 50ul of the enzyme substrate to each well • Wait 5 minutes • positive samples • will begin to turn blue

  20. What Reagents Are We Using? • Purified Antigen: Chicken gamma globulin • Primary antibody (Serum Samples): Polyclonal anti-chicken antibody made by rabbits • Secondary antibody (enzyme-linked): Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish peroxidase (HRP) • Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless solution that when oxidized by HRP turns blue

  21. Results?????

  22. Any Questions?

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