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IMMUNOLOGICAL METHODS

IMMUNOLOGICAL METHODS. Dr. Gülderen Yanıkkaya Demirel. OUTLINE of THIS LECTURE. Definition of Immunology Innate and Adaptive Immunity Antigen/Antibody Immunological Methods. Innate Immunity. Ancient system - present in invertebrates na ï ve, immediate, everyday immunity

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IMMUNOLOGICAL METHODS

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  1. IMMUNOLOGICAL METHODS Dr. Gülderen Yanıkkaya Demirel

  2. OUTLINE of THIS LECTURE • Definition of Immunology • Innate and Adaptive Immunity • Antigen/Antibody • Immunological Methods

  3. Innate Immunity • Ancient system - present in invertebrates • naïve, immediate, everyday immunity • Molecules recognize common features of pathogens • Lectin • Phagocytes, large lymphocytes (NK cells) • Complement

  4. Adaptive Immunity • Newer system - present in fish, birds, human • specialized, late, long term immunity • Molecules recognize specific features of pathogens • Antibodies • B and T cells - small lymphocytes • Immunological memory

  5. Innate and Adaptive Responses Innate • Pathogen independent • Immediate (hours) Neutrophils Macrophages Mast cells Eosinophils Basophils NK cells “Large Lymphocytes” = NK cells Adaptive • Pathogen-dependent • Slower (days) Dendritic cells B cells T cells (CD4 or CD8) “Small Lymphocytes” = B & T cells Both systems “talk” to each other to modulate response Both systems use leukocytes = white blood cells

  6. IMMUNOLOGICAL METHODSdepend onAg Ab interactions

  7. WHAT IS AN ANTIGEN ? • An antigen is any substance that elicits an immune response and is then capable of binding to the subsequently produced antibodies. • Antigens are generally proteins or polysaccharides, but other substances such as nucleic acids can also be antigens.

  8. How, Why and Where are Antibodies Produced?(Massively condensed version) • Produced in vertebrate cells in response to a ‘non-self’ substance termed an antigen. • The process begins with antigen presenting cells roaming the bloodstream. They engulf ‘non-self’ molecules and then display portions of the engulfed molecules on their outer plasma membranes. • After many recognition steps B-cells are stimulated to produce specific antibodies. • The antibodies then go off and bind to the foreign substance thereby marking the substance for destruction.

  9. Recognition modules of Adaptive immunity B-cell receptor (BCR) T cell receptor (TCR) B cells T cells TCR is membrane bound Antibody is a secreted form of BCR

  10. IMMUNOLOGICAL METHODS • Agglutination/Hemaglutination • Immunoprecipitation • Immunodiffusion • Immunoelectrophoresis • RIA/ELISA • Immunoflorescence • Immunohistochemistry • Flow Cytometry • Molecular Methods • PCR • Sequence Analysis • Immunomics • Gene transfection

  11. Qualitative agglutination test • Ag or Ab Y +  Y Y AGGLUTINATION/HEMAGGLUTINATION • Definition - tests that have as their endpoint the agglutination of a particulate antigen • Agglutinin/hemagglutinin

  12. 1/1024 1/256 1/512 1/128 1/16 1/64 1/32 Pos. 1/8 Neg. 1/4 1/2 Titer Patient 64 1 8 2 512 3 <2 4 32 5 128 6 32 7 4 8 AGGLUTINATION/HEMAGGLUTINATION • Quantitative agglutination test • Titer • Prozone

  13. 1/256 1/512 1/128 1/16 1/64 1/32 1/8 1/4 1/2 AGGLUTINATION/HEMAGGLUTINATION • Definition • Qualitative test • Quantitative test • Applications • Blood typing • Bacterial infections • Fourfold rise in titer • Practical considerations • Easy • Semi-quantitative

  14. Ab in gel Ag Ag Ag Ag Diameter2 Ag Concentration Radial Immunodiffusion • Method • Ab in gel • Ag in a well • Interpretation • Diameter of ring is proportional to the concentration • Quantitative • Ig levels

  15. p. 152

  16. - + Ag Ag Ab Ag Ab Immunoelectrophoresis • Method • Ags are separated by electrophoresis • Interpretation • Precipitin arc represent individual antigens

  17. IMMUNE ELECTROPHORESIS

  18. ELISA – Enzyme Linked Immuno Sorbent Assay

  19. RIA – Radio Immuno Assay

  20. ELISPOT

  21. Fluorochrome Labeled Ab Y Ag Tissue Section Immunofluorescence • Direct • Ab to tissue Ag is labeled with fluorochrome

  22. Fluorochrome Labeled Anti-Ig Unlabeled Ab Y Y Ag Tissue Section Immunofluorescence • Indirect • Ab to tissue Ag is unlabeled • Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. • Qualitative to Semi-Quantitative

  23. ImmunoHistochemistry

  24. ImmunoHistochemistry Arrows indicate localized CD3+ lymphocytes in tissue • Antibodies against surface receptors react with tissue on slide. • Secondary antibody with chromgenic probe recognizes first antibody. • Addition of substrate allows deposition of material and histological detection/localization of cell populations within tissue.

  25. FITC FITC Laser Excitation Fluor Detector Light Scatter Detector Add FITC-antibody specific for CD3+ cells Mixed population containing CD3+ cells CD3+ CD3+ CD3+ CD3+ CD3+ CD3+

  26. p. 163

  27. Immunoelectron microscopy

  28. AUTOANTIBODIES

  29. dsDNA

  30. LYMPHOCYTE PROLIFERATION Reactive Lymphocytes Mitogen (ConA) added Antigen added

  31. MAGNETIC BEAD SORTING p. 160

  32. CELL SORTING

  33. MOLECULAR METHODS

  34. Western blotting

  35. GENE TRANSFECTION

  36. SUGGESTED READING • Abbas Immunology 2012 edition • Kuby’s Immunology • Janeway Immunology • Temel İmmunoloji, İmmun sistemin İşlev ve Bozuklukları, 2007.

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