Antigen antibody
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Antigen antibody

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Antigen antibody Presentation Transcript

  • 1. Antigen-Antibody Interactions: Principles & Applications
    • - a bimolecular association
    • involving various noncovalent interactions
    • Is similar to an enzyme-substrate interactions,
    • but not lead to an irreversible chemical alteration
    Chapter 6
  • 2. FIGURE 6-1
    • Four types of non-covalent forces operates over a very short distance
    • ( generally 1 angstrom )
    • - The interaction depends on a very close fit between the Ab & Ag.
  • 3.
    • Strength of Antigen-Antibody Interactions
    • Cross-Reactivity
    • Precipitation Reactions
    • Agglutination Reactions
    • Radioimmunoassay
    • Enzyme-Linked Immunosorbent Assay
    • Western Blotting
    • Immunoprecipitation
    • Immunofluorescence
    • Flow Cytometry and Fluorescence
    • Alternatives to Antigen-Antibody Reactions
    • Immunoelectron Microscopy
  • 4. 1. Strength of Ag-Ab Interactions
    • Antibody affinity
    • is a quantitative measure of binding strength
    • combined strength of the noncovalent interactions
    • between a binding site on an Ab & monovalent Ag
    • Antibody avidity
    • Incorporates affinity of multiple binding sites
    • True strength of the Ab-Ag interaction within biological systems
    • The interaction at one site will increase the possibility
    • of reaction at a second site
    • High avidity can compensate for low affinity
    • ( secreted pentameric IgM has a higher avidity than IgG )
  • 5. 1. Strength of Ag-Ab Interactions
    • High affinity complexes have high Ka values
    • Very stable complexes have very low values of Kd
    Forward & reverse rate constants ( k1 & k-1) Association & dissociation constants ( Ka & Kd ) for 3 ligand-Ab interaction
  • 6. 1. Strength of Ag-Ab Interactions FIGURE 6-2 (a) Determination of Ab affinity ( Ka ) by equilibrium dialysis. Semipermeable membrane A radioactively labeled ligand that is small to pass through ( haptens, oligopeptides )
  • 7. 1. Strength of Ag-Ab Interactions Plot of concentration of ligand in each compartment with time The difference in the two compartments:
  • 8. 1. Strength of Ag-Ab Interactions FIGURE 6-3 (a) Scatchard plots are based on repeated equilibrium dialyses with a constant concentration of Ab and varying concentration of ligand. ( # 1 Ab has a higher affinity than Ab # 2 ) ( all Ab have the same affinity ) (equals moles of bound ligand / mole Ab ) (The binding sites per Ab ) ( c is the conc. Of free ligand )
  • 9. 1. Strength of Ag-Ab Interactions FIGURE 6-3 (b) Scatchard plots yield a curved line whose slope is constantly changing, if Ab preparation is polyclonal. >>> antiserum # 3 has a higher affinity than #4
  • 10. 2. Cross-Reactivity CROSSREACTIVITY (i) Cowpox antigens in vaccinia virus are cross-reactive to smallpox antigens in variola virus (∵ share similar or identical epitope ) (ii) Rabies & JE vaccine >>> encephalitis ( 뇌염 ) (: rabbit brain antigen contaminated vs human brain Ag ) (iii) Streptococcus pyogenes infection >>> heart & Kidney damage following infection (: cell wall proteins called M antigens vs Myocardial & skeletal muscle proteins ). (iv) Original antigenic sin. - The existence of long-lived lymphocytes & crossreactivity - Vaccination with one strain of flu elicited Ab responses to another flu strain. (v) cross-reacting bacterial Ag vs glycoproteins on RBC)
    • Antibody elicited by one Ag can cross-react with unrelated Ag.
    • occurs if two different Ags share identical or very similar epitope
  • 11. 2. Cross-Reactivity ABO blood types - The antibodies are induced by exposure to cross-reacting microbial antigens present on common intestine bacteria. - ABO blood-group antigens have subtle differences in the terminal residues of the sugars on glyco-proteins in RBC. - Providing the basis for blood typing test in blood transfusion
  • 12. 3. 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 )
  • 13. 3. Precipitation Reactions 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. 3. Precipitation Reactions FIGURE 6-6 (a)
    • Immunoelectrophoresis.
    • an antigen mixture is first electrophoresed to separate its components by charge
    • diffusion & producing lines of precipitation.
  • 15. FIGURE 6-7 Demonstration of humaglutination using Ab against sheep red blood cells (SRBCs): a constant # of SRBCs plus serial two-fold dilutions of anti-SRBC serum 4. Agglutination Reactions
    • visible clumping by interaction between Ab & a particulate antigen suchas RBC,
    • latex beads.
    • -depend on the crosslinking of polyvalent antigens, similar to precipitation rxns
    • (lgM is a good agglutinin)
    • -provide a way to type bacteria with a panel of typing antisera.
    • -routinely performed to type RBCs for blood transfusion.
    + + + (control)
  • 16. FIGURE 6-8 -The original home pregnancy test kit employed hapten inhibition ( agglutination inhibition ) to determine the presence or absence of h uman c horionic g onadotropin (HCG) >>> The kits currently on the market use ELISA-based assays . -Also used to determine the use of illegal drugs, & immunity (Ab) to virus (rubella). 4. Agglutination Reactions
  • 17. Sensitivity of various immunoassays
  • 18. 5. Radioimmuno Assay
    • One of the most sensitive technique for measuring hormones, drugs, & vitamins
    • at conc. Of<0.001 ㎍ / ㎖ first discovered by Dr. Berson & Yalow in 1960
    • (1977 Novel prize to Yalow)
    • - The principle involves competitive binding of radiolabeled Ag and unlabeled Ag
    • to the limited supply of a high affinity Ab.
    FIGURE 6-9 A solid-phase radioimmunoassay (RIA) to detect hepatitis B virus in blood samples & A standard curve to determine the conc. of HBsAg in unknown serum.
  • 19. 6. 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
  • 20. FIGURE 6-11 The ELISPOT assay, a modification of the ELISA assay to determine qucontitatively the # of cells in a population that are producing specific Ab or cytokine. 6. ELISA -> precipitates & forms a spot only on the areas of the well where cytokine-secreting cells had been deposited.
  • 21. FIGURE 6-12 7. 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.
  • 22. FIGURE 6-13 Immunoprecipitates can be collected using magnetic beads coupled to a secondary antibody. 8. Immunoprecipitation
    • Ag-Ab attached to a synthetic bead complex >>> 0
    • labeling Ag with radiolabeled leucine, cysteine, or methionine
    • -> A radiolabeled Ag-Ab complex -> 0 -> SDS•PAGE -> autoradiography
    • - Ag-Ab complex + 2 nd Ab attatched to a synthetic bead or magnetic beads >>> 0 or magnet
    EM showing a cell with magnetic beads attached to its surface via antibodies.
  • 23. 9. Immunofluorescence mIgM-producing B cells indirectly stained with rhodamine-conjugated secondary Ab under a fluorescence microscope . FIGURE 6-14
    • Fluorochromes
    • Fluorescein (490->517nm)
    • Rhodamine (515->546nm)
    • Phycoerythrin
    • : absorb light of one wavelength & emit
    • fluorescence at a longer wavelength than
    • fluorescein.
  • 24. FIGURE 6-15 Separation of fluorochrome-labeled cells with the flow cytometer which uses a laser beam & light detector. : different Ag in different cells / different levels of Ag in the same type of cell -> fluorescence intensity / the size of cells. 10. Flow cytometry & Fluorescence labeled with fluorescein (green) labeled with rhodamine (red) each dot represents a cell (small electrical change) (exciting the fluorochrome) ↓ each droplet (cell) emits the fluorescence
  • 25. 11. Alternalives to Ag-Ab Reactions Ag-Ab-Ab* ->Ag-IgG-A/G* or Ag-Ab-biotin-(a)vidin* ① Protein A (from staphylococcus) & protein G (from streptococcus) - bind to rhe Fc region of lgG molecules (k a ~ 10 8 ) - used to detect lgG molecules in the Ag-Ab complexes - used to isolate lgG molecules in the affinity columns ② Avidin (from egg whites) & streptavidin (from streptomyces avidinii) conjugated with an enzyme, fluorechrome, radioactive label) - bind to biotin (a vitamin) with higher affinity (k a ~ 10 15 ) - Ab can be labeled with (k a ~ 10 18 )
  • 26. FIGURE 6-16
    • An immunoelectronmicrograph of the surface of a B-cell lymphoma was stained with two antibodies (Ab against class II MHC labeled sith 30nm gold particles, & another Ab against class I MHC w/ 15nm gold particles.
    • (The density of class I exceeds that of class II)
    • Electron-dense label (ferritin or colloidal gold) is conjugated to the Fc
    • portion.
    12. Immuno EM. electron-denselabels Absorbs electrons.
  • 27. CLINICAL FOCUS Distribution of selected markers on some leukemic cell types (leukemia can wise at any maturational stage of any one of the hematopoietic lineages) -> Immuno phenotyping: the determination of the profile of selected cell- surface markers displayed by the leukemic cell, using “flow cytometry & mAb” [: a cute l ymphocytic l eukemia] [: c hronic l ymphocytic l eukemia]