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Ag-Ab reactions Tests for Ag-Ab reactions

Ag-Ab reactions Tests for Ag-Ab reactions. http://www.med.sc.edu:85/chime2/lyso-abfr.htm. Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000. Nature of Ag/Ab Reactions. Lock and Key Concept. Non-covalent Bonds. Hydrogen bonds Electrostatic bonds

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Ag-Ab reactions Tests for Ag-Ab reactions

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  1. Ag-Ab reactionsTests for Ag-Ab reactions

  2. http://www.med.sc.edu:85/chime2/lyso-abfr.htm Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 Nature of Ag/Ab Reactions • Lock and Key Concept • Non-covalent Bonds • Hydrogen bonds • Electrostatic bonds • Van der Waal forces • Hydrophobic bonds • Multiple Bonds • Reversible

  3. Low Affinity High Affinity Ab Ab Ag Ag Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site Affinity =  attractive and repulsive forces

  4. [Ag-Ab] Keq = [Ag] x [Ab] Calculation of Affinity Ag + Ab  Ag-Ab Applying the Law of Mass Action:

  5. Y Y Y Y Y Y Y 104 106 1010 Keq = Avidity Affinity Avidity Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs

  6. 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.

  7. Cross reactions Anti-A Ab Anti-A Ab Anti-A Ab Ag B Ag C Shared epitope Similar epitope Ag A 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

  8. Ab excess Ag excess Equivalence – Lattice formation Factors Affecting Measurement of Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag

  9. 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

  10. Agglutination Tests Lattice Formation

  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. Y Y +  Y Passive Agglutination/Hemagglutination • Definition - agglutination test done with a soluble antigen coated onto a particle • Applications • Measurement of antibodies to soluble antigens

  15. 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 • Incomplete Ab • Direct Coombs Test • Detects antibodies on erythrocytes

  16. Step 1 Y +  Y Y Y Y Target RBCs Patient’s Serum Y Y Y Step 2 Y Y Y Y Y Y Y Y Y Y Y Y +  Y Y Y Y Coombs Reagent (Antiglobulin) Coombs (Antiglobulin)Tests • Indirect Coombs Test • Detects anti-erythrocyte antibodies in serum

  17. Coombs (Antiglobulin)Tests • Applications • Detection of anti-Rh Ab • Autoimmune hemolytic anemia

  18. Prior to Test Y Y +  Y Y Test Y + +  Y Patient’s sample Agglutination/Hemagglutination Inhibition • Definition - test based on the inhibition of agglutination due to competition with a soluble Ag

  19. Agglutination/Hemagglutination Inhibition • Definition • Applications • Measurement of soluble Ag • Practical considerations • Same as agglutination test

  20. Precipitation Tests Lattice Formation

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

  22. - + Ag Ag Ab Ag Ab Immunoelectrophoresis • Ab is placed in trough cut in the agar • Method • Ags are separated by electrophoresis • Interpretation • Precipitin arc represent individual antigens

  23. Immunoelectrophoresis • Method • Interpretation • Qualitative • Relative concentration

  24. - + Ab Ag Countercurrent electrophoresis • Method • Ag and Ab migrate toward each other by electrophoresis • Used only when Ag and Ab have opposite charges • Qualitative • Rapid

  25. Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required

  26. Prior to Test Y Y +  Labeled Ag Test Y Y + + +  Labeled Ag Patient’s sample 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

  27. Solid Phase Solid Phase Test Y Y + + +  Labeled Ag Patient’s sample Competitive RIA/ELISA for Ag • Method cont. • Determine amount of labeled Ag bound to Ab • NH4SO4 •  anti-Ig • Immobilize the Ab • Concentration determined from a standard curve using known amounts of unlabeled Ag • Quantitative • Most sensitive test

  28. Labeled Anti-Ig Ab in Patient’s sample Y Y Ag Immobilized 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

  29. Labeled Ab Ag in Patient’s sample Y Ag Y Immobilized Solid Phase 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

  30. Tests for Cell Associated Antigens Lattice formation not required

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

  32. 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

  33. Flow Tip FL Detector Light Scatter Detector Laser Immunofluorescence • Flow Cytometry • Cells in suspension are labeld with fluorescent tag • Direct or Indirect Fluorescence • Cells analyzed on a flow cytometer

  34. Two Parameter Histogram Green Fluorescence Intensity Red Fluorescence Intensity Immunofluorescence • Flow Cytometry cont. • Data displayed One Parameter Histogram Unstained cells FITC-labeled cells Number of Cells Green Fluorescence Intensity

  35. Assays Based on Complement Lattice formation not required

  36. No Ag Ag Patient’s serum Y Y Y Y Y Y Y Y Y Y Complement Fixation • Methodology • Standard amount of complement is added • Ag mixed with test serum to be assayed for Ab • Erythrocytes coated with Abs is added • Amount of erythrocyte lysis is determined Ag Ag

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