Elis as 1

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  • Elis as 1

    1. 1. ELISAsEnzyme Linked Immunosorbent Assays
    2. 2. Antibody-Antigen ReactionsApplications Agglutination reactions Precipitation reactions Western blotting/immunoblotting Direct and indirect immunofluorescence Flow Cytometry ELISAs
    3. 3. What the ELISA tells us
    4. 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. 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. 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. 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. 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. 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. 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. 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. 12. The Indirect ELISA Measures antibody
    13. 13. The Indirect ELISA
    14. 14. The Indirect ELISA
    15. 15. The Indirect ELISA
    16. 16. The Indirect ELISA
    17. 17. The Indirect ELISA
    18. 18. The Indirect ELISA
    19. 19. The Sandwich ELISA Measures antigen
    20. 20. The Sandwich ELISA
    21. 21. The Sandwich ELISA
    22. 22. The Sandwich ELISA
    23. 23. The Sandwich ELISA
    24. 24. The Sandwich ELISA
    25. 25. The Sandwich ELISA
    26. 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. 27. The Competative ELISA
    28. 28. The Competative ELISA
    29. 29. The Competative ELISA
    30. 30. The Competative ELISA
    31. 31. The Competative ELISA
    32. 32. The Competative ELISA
    33. 33. The Competative ELISA
    34. 34. The Competative ELISA
    35. 35. Add capture Wash antibody Block Wash Dilute Aliquot AliquotStandard Standard Unknown Incubate

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