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basics of immunology

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  1. 1. Antigen-Antibody Interactions: Principles & Applications --by K.R.Deepthi <ul><li>- A bimolecular association </li></ul><ul><li>involving various noncovalent interactions </li></ul><ul><li>Is similar to an enzyme-substrate interactions, </li></ul><ul><li>but not lead to an irreversible chemical alteration </li></ul>
  2. 2. Nature of Ag/Ab Reactions <ul><li>Four types of non-covalent forces operates over a very short distance </li></ul><ul><li>( generally 1 angstrom ) </li></ul>
  3. 3. <ul><li>Strength of Antigen-Antibody Interactions </li></ul><ul><li>Precipitation Reactions </li></ul><ul><li>Agglutination Reactions </li></ul><ul><li>Immunodiffusion </li></ul><ul><li>Radioimmunoassay </li></ul><ul><li>Enzyme-Linked Immunosorbent Assay </li></ul><ul><li>Western Blotting/immunoblotting </li></ul><ul><li>Immunofluorescence </li></ul><ul><li>Immunohistocompatibility </li></ul><ul><li>Localization of cells in tissue immunoblotting. </li></ul>contents:
  4. 4. Structure of an antibody
  5. 5. Affinity =  attractive and repulsive forces Affinity <ul><li>Strength of the reaction between a single antigenic determinant and a single Ab combining site </li></ul>Ab Ag High Affinity Ab Ag Low Affinity
  6. 6. Calculation of Affinity Ag + Ab  Ag-Ab Applying the Law of Mass Action: K eq = [Ag-Ab] [Ag] x [Ab]
  7. 7. Avidity <ul><li>The overall strength of binding between an Ag with many determinants and multivalent Abs </li></ul>Y K eq = 10 4 Affinity Y 10 6 Avidity Y Y Y Y Y 10 10 Avidity
  8. 8. Specificity <ul><li>The ability of an individual antibody combining site to react with only one antigenic determinant. </li></ul>
  9. 9. Cross Reactivity <ul><li>The ability of an individual Ab combining site to react with more than one antigenic determinant. </li></ul><ul><li>The ability of a population of Ab molecules to react with more than one Ag </li></ul>Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope Anti-A Ab Ag C Similar epitope Cross reactions
  10. 10. Factors Affecting Measurement of Ag/Ab Reactions <ul><li>Affinity </li></ul><ul><li>Avidity </li></ul><ul><li>Ag:Ab ratio </li></ul><ul><li>Physical form of Ag </li></ul>Ab excess Ag excess Equivalence – Lattice formation
  11. 11. Precipitation Reactions Precipitation reactions in fluids yield a precipitin curve. FIGURE 6-4 ( Lattices or large aggregates ) ( no precipitate is formed if an Ag contains only a single copy of each epitope )
  12. 12. Radial Immunodiffusion (Mancini) <ul><li>Interpretation </li></ul><ul><ul><li>Diameter of ring is proportional to the concentration </li></ul></ul><ul><li>Quantitative </li></ul><ul><ul><li>Ig levels </li></ul></ul><ul><li>Method </li></ul><ul><ul><li>Ab in gel </li></ul></ul><ul><ul><li>Ag in a well </li></ul></ul>Ag Concentration Diameter 2 Ag Ag Ag Ag Ab in gel
  13. 13. FIGURE 6-5 Diagrammatic representation of radial & double immunodiffusion. : precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess. in the Ab-containing semisolid medium The region of equivalence -> The area is proportional to the conc. of Ag.
  14. 14. Precipitation Reactions(immunoelectrophorosis) FIGURE 6-6 (a) <ul><li>Immunoelectrophoresis. </li></ul><ul><li>an antigen mixture is first electrophoresed to separate its components by charge </li></ul><ul><li>diffusion & producing lines of precipitation. </li></ul>
  15. 15. Countercurrent electrophoresis <ul><li>Method </li></ul><ul><ul><li>Ag and Ab migrate toward each other by electrophoresis </li></ul></ul><ul><ul><li>Used only when Ag and Ab have opposite charges </li></ul></ul><ul><li>Qualitative </li></ul><ul><ul><li>Rapid </li></ul></ul>Ag Ab - +
  16. 16. Agglutination/Hemagglutination <ul><li>Definition - tests that have as their endpoint the agglutination of a particulate antigen </li></ul><ul><ul><li>Agglutinin/hemagglutinin </li></ul></ul>Y Y Y +  <ul><li>Qualitative agglutination test </li></ul><ul><ul><li>Ag or Ab </li></ul></ul>
  17. 17. FIGURE 6-7 Demonstration of hemagglutination using Ab against sheep red blood cells (SRBCs). Agglutination Reactions <ul><li>visible clumping by interaction between Ab & a particulate antigen such as RBC, </li></ul><ul><li>latex beads. </li></ul><ul><li>-routinely performed to type RBCs for blood transfusion. </li></ul>+ + + (control)
  18. 18. RIA
  19. 19. <ul><li>From these data, a standard binding curve, like th e one shown in red, can be drawn. </li></ul>RIA
  20. 20. RIA <ul><li>Radioimmunoassay is widely-used because of its great sensitivity. </li></ul><ul><li>Using antibodies of high affinity, it is possible to detect a few picograms (10 −12 g) of antigen in the tube. </li></ul><ul><li>The greater the specificity of the antiserum, the greater the specificity of the assay </li></ul>
  21. 21. ELISA FIGURE 6-10 Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to RIA except using an Enzyme (alkaline ⓟ, horseradish peroxidase, & β-galactosidase) : safer & less costly. to detect Ab (HIV, HCV) to detect Ag to detect Ag
  22. 22. FIGURE 6-12 Western blotting : separates the components according to their molecular weight. : the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current. : probed with Ab & then radiolabeled or enzyme-linked 2 nd Ab. : a position is visualized by means of an ELISA reaction.
  23. 23. Immunofluorescence mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope . FIGURE 6-14 <ul><li>Fluorochromes </li></ul><ul><li>Fluorescein (490->517nm) </li></ul><ul><li>Rhodamine (515->546nm) </li></ul><ul><li>Phycoerythrin </li></ul><ul><li>: absorb light of one wavelength & emit </li></ul><ul><li>fluorescence at a longer wavelength than </li></ul><ul><li>fluorescein. </li></ul>
  24. 24. Localization of cells in tissue immunoblotting <ul><li>  Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots and dicots. </li></ul><ul><li>however, very little is known about the tissue and cell type localization as well as the physiological function(s) of these oxygen-binding proteins. </li></ul><ul><li>The immunodetection and immunolocalization of ns-Hbs in rice ( Oryza sativa L.) by Western blotting and in situ confocal laser scanning techniques. Ns-Hbs were detected in soluble extracts of different tissues from the developing rice seedling by immunoblotting. Levels of ns-Hbs increased in the germinating seed for the first six days following imbibition and remained relatively constant thereafter. </li></ul>
  25. 25. <ul><li>It is similar to grafting </li></ul>Immunohistocompatibility