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Immunology Session 3 U se of antibodies (PART 1)

Immunology Session 3 U se of antibodies (PART 1). Immunology Tutorial Outline. 1 Revision of key concepts 2 Radioimmunoassays 3 Single radial immunodiffusion. 1 revision Antibody structure (p295). Precipitating and Purifying Antibodies (p309-314).

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Immunology Session 3 U se of antibodies (PART 1)

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  1. ImmunologySession 3Use of antibodies (PART 1)

  2. ImmunologyTutorial Outline 1 Revision of key concepts 2 Radioimmunoassays 3 Single radial immunodiffusion

  3. 1 revision Antibody structure (p295)

  4. Precipitating and Purifying Antibodies (p309-314) When a lot of salt, (eg ammonium sulfate), is added to the protein solution, the salt ions attract water molecules away from the protein, partly since salt ions have a greater charge density than proteins. As the salt is added These ions bind water molecules, protein molecules are forced to interact with themselves and begin to aggregate and will precipitate

  5. 1 revision Precipitating and Purifying Antibodies (p310) Read over the following methods: Gel filtration chromatography Ion Exchange chromatography Affinity chromatography CLICK HERE to see THERMO SCIENTIFIC Production and Use of Antibodies

  6. 1 revision Precipitating and Purifying Antibodies (p309) Affinity chromatography

  7. 1 revision Immunoprecipitiation techniques (p315) Most antibodies can precipitate most antigens from solution (especially if they have many epitopes). At the right concentrations, the antibody-antigen lattice that forms is too large to remain in solution…. This is a narrow range called the: ZONE Of EQUIVALENCE

  8. 1 revision Immunoprecipitiation techniques (p316)

  9. 1 revision Immunoprecipitiation techniques (p315)

  10. 1 revision Immunoprecipitiation techniques (p315) This property of antibody-antigen precipitation can be used in a wide range of techniques including: Radioimmunoassays Single radial immunodiffusion Double Diffusion Immunoelectrophoresis

  11. Radioimmunoassays Particularly useful in endocrinology – hormone assays…. based on competition between unlabelled antigen and a finite amount of radiolabelled antigen for an antibody in FIXED, LIMITED amounts Principle of standard curve construction: 4Ag* + 4AB g4Ag*AB 4Ag + 4Ag * + 4AB g2Ag*AB + 2AgAB + 2Ag* +2Ag 12Ag + 4Ag * + 4AB gAg*AB + 3AgAB + 3Ag* + 9Ag

  12. Radioimmunoassays Principle of standard curve construction 4Ag* + 4AB g4Ag*AB 100% bound 4Ag + 4Ag * + 4AB g 2Ag*AB + 2AgAB + 2Ag* +2Ag 50% bound 12Ag + 4Ag * + 4AB g Ag*AB + 3AgAB + 3Ag* + 9Ag 25% bound Bound labelled antigen %

  13. Radioimmunoassays Finding the unknown…. 4Ag* + 4AB g4Ag*AB 100% bound Add X amount of antigen (from the sample) ....a certain amount of bound radioisotope eg 50% Whats the value of X? Bound labelled antigen %

  14. Radioimmunoassays http://www.phoenixpeptide.com/catalog/product_info.php?products_id=4409

  15. 1 revision Immunoprecipitiation techniques in gels (p316) Principle When antigens diffuse from a homogenous solution into an agar gel the concentration fals from a maximum at the solution/gel interface to zero at the leading edge of the region penetrated. Somewhere along this concentration gradient will be the ZONE OF EQUILIVALENCE…and the precipitation which occurs here can be used to calculate the concentration in the original solution

  16. Immunoprecipitiation Techniques

  17. Immunoprecipitiation Techniques

  18. Immunoprecipitiation Techniques

  19. Immunoprecipitiation Techniques

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