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8 ag ab reactions07
 

8 ag ab reactions07

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    8 ag ab reactions07 8 ag ab reactions07 Presentation Transcript

    • Ag-Ab reactions Tests for Ag-Ab reactions
    • Nature of Ag/Ab Reactions
      • Lock and Key Concept
      • Non-covalent Bonds
      • Hydrogen bonds
      • Electrostatic bonds
      • Van der Waal forces
      • Hydrophobic bonds
      • Reversible
      • Multiple Bonds
      Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 http://www.med.sc.edu:85/chime2/lyso-abfr.htm
    • Affinity = ∑ attractive and repulsive forces Affinity
      • Strength of the reaction between a single antigenic determinant and a single Ab combining site
      Ab Ag High Affinity Ab Ag Low Affinity
    • Calculation of Affinity Ag + Ab  Ag-Ab Applying the Law of Mass Action: K eq = [Ag-Ab] [Ag] x [Ab]
    • Avidity
      • The overall strength of binding between an Ag with many determinants and multivalent Abs
      Y K eq = 10 4 Affinity Y 10 6 Avidity Y Y Y Y Y 10 10 Avidity
    • Specificity
      • The ability of an individual antibody combining site to react with only one antigenic determinant.
      • The ability of a population of antibody molecules to react with only one antigen.
    • Cross Reactivity
      • The ability of an individual Ab combining site to react with more than one antigenic determinant.
      • The ability of a population of Ab molecules to react with more than one Ag
      Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope Anti-A Ab Ag C Similar epitope Cross reactions
    • Factors Affecting Measurement of Ag/Ab Reactions
      • Affinity
      • Avidity
      • Ag:Ab ratio
      • Physical form of Ag
      Ab excess Ag excess Equivalence – Lattice formation
    • Tests Based on Ag/Ab Reactions
      • All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes
      • All tests based on Ag/Ab reactions can be used to detect either Ag or Ab
    • Agglutination Tests Lattice Formation
    • Agglutination/Hemagglutination
      • Definition - tests that have as their endpoint the agglutination of a particulate antigen
        • Agglutinin/hemagglutinin
      Y Y Y + 
      • Qualitative agglutination test
        • Ag or Ab
    • Agglutination/Hemagglutination
      • Quantitative agglutination test
        • Titer
        • Prozone
      1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 32 4 Patient 1 2 3 4 5 6 7 8
    • Agglutination/Hemagglutination
      • Definition
      • Qualitative test
      • Quantitative test
      • Applications
        • Blood typing
        • Bacterial infections
          • Fourfold rise in titer
      • Practical considerations
        • Easy
        • Semi-quantitative
      1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512
    • Passive Agglutination/Hemagglutination
      • Definition - agglutination test done with a soluble antigen coated onto a particle
      • Applications
        • Measurement of antibodies to soluble antigens
      Y Y Y + 
    • Coombs (Antiglobulin)Tests
      • Incomplete Ab
      • Direct Coombs Test
        • Detects antibodies on erythrocytes
      +  Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin)
    • Coombs (Antiglobulin)Tests
      • Indirect Coombs Test
        • Detects anti-erythrocyte antibodies in serum
      Y Y Y Y Y Patient’s Serum Target RBCs +  Step 1 +  Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagent (Antiglobulin) Step 2
    • Coombs (Antiglobulin)Tests
      • Applications
        • Detection of anti-Rh Ab
        • Autoimmune hemolytic anemia
    • Agglutination/Hemagglutination Inhibition
      • Definition - test based on the inhibition of agglutination due to competition with a soluble Ag
      Y Y Y +  Prior to Test Y +  Y Y + Test Patient’s sample
    • Agglutination/Hemagglutination Inhibition
      • Applications
        • Measurement of soluble Ag
      • Practical considerations
        • Same as agglutination test
      • Definition
    • Precipitation Tests Lattice Formation
    • Radial Immunodiffusion (Mancini)
      • Interpretation
        • Diameter of ring is proportional to the concentration
      • Quantitative
        • Ig levels
      • Method
        • Ab in gel
        • Ag in a well
      Ag Concentration Diameter 2 Ag Ag Ag Ag Ab in gel
    • I mmunoelectrophoresis
      • Method
        • Ags are separated by electrophoresis
      • Interpretation
        • Precipitin arc represent individual antigens
        • Ab is placed in trough cut in the agar
      Ag - + Ag Ab Ag Ab
    • I mmunoelectrophoresis
      • Method
      • Interpretation
      • Qualitative
        • Relative concentration
    • Countercurrent electrophoresis
      • Method
        • Ag and Ab migrate toward each other by electrophoresis
        • Used only when Ag and Ab have opposite charges
      • Qualitative
        • Rapid
      Ag Ab - +
    • Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required
    • Competitive RIA/ELISA for Ag
      • Method
        • Determine amount of Ab needed to bind to a known amount of labeled Ag
        • Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
      Y Y +  Prior to Test Labeled Ag Y Y +  Test + Patient’s sample Labeled Ag +
    • Competitive RIA/ELISA for Ag
      • Method cont.
        • Determine amount of labeled Ag bound to Ab
          • ↓ NH 4 SO 4
          • ↓ anti-Ig
          • Immobilize the Ab
      • Quantitative
        • Most sensitive test
        • Concentration determined from a standard curve using known amounts of unlabeled Ag
      Y Y +  Test + Patient’s sample Labeled Ag + Solid Phase Solid Phase
    • Solid Phase Non-Competitive RIA/ELISA
      • Ab detection
        • Immobilize Ag
        • Incubate with sample
        • Add labeled anti-Ig
        • Amount of labeled Ab bound is proportional to amount of Ab in the sample
      • Quantitative
      Solid Phase Y Ag Immobilized Y Ab in Patient’s sample Labeled Anti-Ig
    • Solid Phase Non-Competitive RIA/ELISA
      • Ag detection
        • Immobilize Ab
        • Incubate with sample
        • Add labeled antibody
        • Amount of labeled Ab bound is proportional to the amount of Ag in the sample
      • Quantitative
      Solid Phase Y Ag Immobilized Y Ag in Patient’s sample Labeled Ab
    • Tests for Cell Associated Antigens Lattice formation not required
    • Immunofluorescence
      • Direct
        • Ab to tissue Ag is labeled with fluorochrome
      Ag Y Fluorochrome Labeled Ab 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
      Ag Y Y Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab
    • Immunofluorescence
      • Flow Cytometry
        • Cells in suspension are labeled with fluorescent tag
          • Direct or Indirect Fluorescence
        • Cells analyzed on a flow cytometer
      Flow Tip Laser FL Detector Light Scatter Detector
    • Immunofluorescence
      • Flow Cytometry cont.
        • Data displayed
      Green Fluorescence Intensity Number of Cells Unstained cells FITC-labeled cells One Parameter Histogram Red Fluorescence Intensity Green Fluorescence Intensity Two Parameter Histogram
    • Assays Based on Complement Lattice formation not required
    • Complement Fixation
        • Ag mixed with test serum to be assayed for Ab
        • Standard amount of complement is added
        • Erythrocytes coated with Abs is added
        • Amount of erythrocyte lysis is determined
      Ag Ag
      • Methodology
      Y Y Patient’s serum Y Y Y Y Y Y Y Y Ag No Ag