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9th SEMINAR

9th SEMINAR. LABORATORY METHODS BASED ON ANTIGEN-ANTIBODY INTERACTIONS II. THE SENSITIVITY OF IMMUNOASSAYS. Sensitive methods: precise expensive usually used for verification. Less sensitive methods: give semiquantitative results cheap usually used for screening. FLOW CYTOMETRY.

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9th SEMINAR

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  1. 9th SEMINAR LABORATORY METHODS BASED ON ANTIGEN-ANTIBODY INTERACTIONSII

  2. THE SENSITIVITY OF IMMUNOASSAYS • Sensitive methods: • precise • expensive • usually used for verification • Less sensitive methods: • give semiquantitative results • cheap • usually used for screening

  3. FLOW CYTOMETRY An immunofluorescent method that is similar to immunohistochemistry but gives rather quantitative than qualitative results • Investigation of cells travelling in a flow with high velocity • Detects light scatter of particles and fluorescence intensity of the labeled cells Enormous number of cells can be investigated in short period of time

  4. ADVANTAGES OF FLOW CYTOMETRY • Most cells can be easily suspended and labeled by fluorescent cellsurface marker-specific antibodies • The cells’ light scatter and immunofluorescent properties can be analyzed statistically • Rare cell populations can be identified and examined (e.g. antigen specific lymphocytes) • The method provides both qualitative and quantitative data: presence of antigen and the expression levels of these antigensChanges in the expression of certain molecules can be followed after different treatments (e.g. cell activation, disease progression)

  5. BASIC STRUCTURE OF THE FLOW CELL fluid flow sheath fluid Forward angle light scatter sensor (FSC) Laser represents size Side light scatter (SSC) represents granularity and fluorescence detectors

  6. DENSITY PLOT OF PERIPHERAL BLOOD CELLS AFTER LYISING RED BLOOD CELLSDifferentcell types  characteristic light scattering granulocytes side scatter (SSC) (granularity) monocytes lymphocytes forward scatter (FSC) (size)

  7. CELL TYPES, DIFFERENTIATION STAGES CAN BE IDENTIFIED USING A COMBINATION OF CELL SURFACE MARKERS Used in diagnostics: • ratio of different cell types • altered expression of cell surface markers Examples: • Inflammatory processes – increased neutrophil numbers • increase of CD5+ B cells – typical for some B cell leukemias • HIV progression – decrease of CD4+ T cell count In normal case CD4+ : CD8+ = 1.6 Normal CD4+ T cell count = 600 – 1400/l AIDS = CD4+ T cell count <200/l

  8. Fluorescent microscopy: IMMUNOPHENOTYPING BY FLOW CYTOMETRY Example:Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio (e.g. monitoring AIDS progression) FITC labeled anti-CD4 antibody(α-CD4-FITC) PE labeled anti-CD8 antibody (α-CD8-PE) Labeling: Lymphocytes in the peripheral blood sample: B NK Th Tc

  9. detecting CD4-FITC labeled (TH) cell Th increasing fluorescence intensity a dot representing a CD4+ CD8- cell microscopy: high velocity flow stream (in cuvette or stream in air) signal processing unit screen detector CD8 PE focused laser beam CD4 FITC

  10. detecting the PE labeled cell (CD8-PE) Tc increasing fluorescence intensity signal processing unit detector CD8 PE CD4 FITC

  11. detecting an unlabeled cell (e.g. B cell) by autofluorescence B increasing fluorescence intensity microscopy: dim (autofluorescent) cell Signal processing unit detector CD8 PE CD4 FITC

  12. Signal processing unit 18% 0% CD8 PE quadrant statistics 44% 38% CD4 FITC

  13. dot-plot contour- plot density- plot GRAPHICAL REPRESENTATIONSI

  14. GRAPHICAL REPRESENTATIONS II Histogramm homogenous cell population has normal distribution (Gaussian) Numeral intensity values: ~ 7 ~ 1300

  15. THE SENSITIVITY OF IMMUNOASSAYS • Sensitive methods: • precise • expensive • usually used for verification • Less sensitive methods: • give semiquantitative results • cheap • usually used for screening

  16. FORMATION OF IMMUNECOMPLEXES Immune complexes are rapidly formed in solutions. In a steady-state equilibrium, determined by the dissociation constant (Kd), they may form precipitates by secondary interactions

  17. PRECIPITATION OF IMMUNE COMPLEXES antibody excess EQUIVALENCE antigen excess precipitated proteins Increasing antigen amounts antibody excess antigen excess precipitated proteins Equivalent amount of Ab and Ag will lead to a visible precipitation Excess in either Ab or Ag will lead to soluble complexes Increasing antibody amounts

  18. IMMUNODIFFUSION METHODS I Mancini method / Radial immunodiffusion 2D simple immodiffusion Antigen Antibody In gel Antigen Antibody The size of the precipitation ring is relative to the concentration of the antigen; higher concentrations of antigens will diffuse further until they reach the equilibrium Applied for quantitative determination of viral antigens or virus specific antibodies in the serum

  19. La Sm Ro RNP Jo1 Scl-70 patient’s serum sample IMMUNODIFFUSION METHODS II Ouchterlony method 2D double immunodiffusion antigen antibody antigen antibody Gold standard method for detection of Extractable Nuclear Antigen (ENA)-specific autoantibodies ENAs: e.g. Ro, La, Sm, RNP, Scl-70, Jo1 Autoantigens located in the nuclei of cells. In case of systemic autoimmune disorders autoreactive B-cells specific to these antigens get activated and continuously produce antibodies against their targets.

  20. AGGLUTINATION REACTIONS • Agglutination: The clumping of cells such as bacteria or red blood cells in the presence of antibodies. • It differs from precipitation only in size – here the antigen carrying substances are cells, or colloidal structures. • Hemagglutination: When the particles involved are RBCs.

  21. TYPES AND USES OF AGGLUTINATION REACTIONS • DIRECT AGGLUTINATION Aggregation followed by antibody binding (mostly IgM) (AB0 blood group antigens) • INDIRECT AGGLUTINATION Aggregation is achieved with a secondary antibody recognizing the Fc region of the primary antibody (mostly IgG) (Rh blood group- D antigen) • PASSIVE AGGLUTINATION RBCs or latex particles (latex beads) to which haptensor protein antigensartificially are coupled, aggregate after the addition of the specific antibodies (RF- Rheumatoid Factor detection)

  22. DIRECT AGGLUTINATION A, B or both AB antigens on the surface of RBCs will bind to the appropriate anti-A, Anti-B antibodies and agglutinate • AB0 blood group typing • Detecting the presence of an antigen

  23. DONORS AND RECIPIENTS FOR BLOOD TRANSFUSION Only theoretically, recipients must receive their own type of blood!

  24. QUANTITATIVE HAEMAGGLUTINATION TEST Quantitative detection of antibodies (antibody-titer) specific to a given antigen. The antigen ispresent on the surface of RBCs / bacteria, or fixed to the surface of RBCs / latex beads.

  25. INDIRECT AGGLUTINATION Primary AB Secondary AB • Rh blood group determination • Diagnosis of autoimmune hemolytic anemia • Cross matching donor and recipient; ABO and Rh compatibility

  26. Rh prophylaxis EFFECTS OF AGGLUTINATION IN VIVO ABO incompatibility intravascular hemolysis (complement mediated hemolysis) Rh incompatibility hemolytic disease of the newborn (erythroblastosisfetalis) (opsonisation of red blood cells, which are then phagocytosed by macrophages and granulocytes) Administration of Anti-D IgG vaccine within 72 hours after birth of the first Rh-incompatible fetus prevents production of maternal antibodies.

  27. Erythropoiesis in lung Erythroblasts in bone marrow PATHOLOGICAL CONSEQUENCES OF PLACENTAL TRANSPORT OF IgG (hemolytic disease of the newborn) anti-Rh

  28. Both Coombs tests are indirect agglutinations because the usage of secondary antibodies Direct Coombs test – e.g. detection of autoimmune hemolitic anemia Indirect Coombs test – e.g. detection of minor blood group incompatibility

  29. PASSIVE AGGLUTINATION Fixating antigens on the surface of latex beads / RBCs and mixing them with the patient’s serum Rheuma factor is self IgG/IgM reactive IgM produced by autoreactive plasma cells • Latex agglutination test for Rheumatoid Factor in Rheumatoid Arthritis

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