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Elis as 1
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Elis as 1

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  • Transcript

    • 1. ELISAsEnzyme Linked Immunosorbent Assays
    • 2. Antibody-Antigen ReactionsApplications Agglutination reactions Precipitation reactions Western blotting/immunoblotting Direct and indirect immunofluorescence Flow Cytometry ELISAs
    • 3. What the ELISA tells us
    • 4. What the ELISA tells us The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding.
    • 5. What the ELISA tells us The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding.
    • 6. What the ELISA tells us The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding. Depending on what variation is used, ELISAs will detect antigen or antibody.
    • 7. Applications of ELISAAntigens detected by ELISAs include: hormones enzymes microbial antigens illicit drugsAntibodies detected by ELISAs include: antibodies in body fluids antibodies in tissue culture supernatants anti-HIV in the screening test for HIV infection Anti-West Nile in the screening for West Nile Virus
    • 8. Advantages of the ELISA methodThe ELISA is probably the most commonlyused immunological assay because of its: versatility sensitivity (ability to detect small amounts of antigen or antibody) specificity (ability to discriminate between closely related but antigenically different molecules)
    • 9. What is needed to perform the assay  Purified antigen (if you want to detect or quantify antibody).  Purified antibody (if you want to detect or quantify antigen).  Standard solutions (positive and negative controls).  Sample to be tested.  Microtiter dishes: plastic trays with small wells in which the assay is done.  Wash fluid (buffer).  Enzyme-labeled antibody and enzyme substrate.  ELISA reader (spectrophotometer) for quantitative measurements.
    • 10. What we are using Capture antibody: α-IL-2 Purified antigen: IL-2 Detection antibody: HRP conjugated α-IL-2 Wash buffer: PBS-T Enzyme substrate: Stabilized 3,3’, 5,5’ Tetramethylbenzidine
    • 11. How to interpret the results The amount of colored product is proportional to the amount of enzyme-linked antibody that binds, which is directly related to the amount of antibody that was present to bind antigen or antigen that was present to bind antibody. If known amounts of antigen or antibody are added, a standard curve can be constructed which will allow the amount of unknown antigen or antibody to be determined.
    • 12. The Indirect ELISA Measures antibody
    • 13. The Indirect ELISA
    • 14. The Indirect ELISA
    • 15. The Indirect ELISA
    • 16. The Indirect ELISA
    • 17. The Indirect ELISA
    • 18. The Indirect ELISA
    • 19. The Sandwich ELISA Measures antigen
    • 20. The Sandwich ELISA
    • 21. The Sandwich ELISA
    • 22. The Sandwich ELISA
    • 23. The Sandwich ELISA
    • 24. The Sandwich ELISA
    • 25. The Sandwich ELISA
    • 26. The Competitive ELISA This assay is based on the competitive binding technique in which antigen present in a sample competes with a fixed amount of enzyme conjugate for binding sites on an antibody- coated plate. The extent of color development is inversely proportional to the amount of antigen in the sample. Measures antigen
    • 27. The Competative ELISA
    • 28. The Competative ELISA
    • 29. The Competative ELISA
    • 30. The Competative ELISA
    • 31. The Competative ELISA
    • 32. The Competative ELISA
    • 33. The Competative ELISA
    • 34. The Competative ELISA
    • 35. Add capture Wash antibody Block Wash Dilute Aliquot AliquotStandard Standard Unknown Incubate

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