s2004assays.ppt
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  • 1. Immunological assays Antigen-antibody interactions Antibody-based assays
  • 2. Affinity and specificity for antigen Assays detect presence of antigen or antibody diagnosis monitoring humoral immune response therapeutics analysis of interesting molecules
  • 3. Nature of antigen-antibody interaction p. 138
  • 4. Affinity- strength of interaction between one Ag-binding site and one epitope Measured by various methods including equilibrium dialysis, competition assays, microchips (e.g., Biacor) Association constants can be calculated
  • 5. Avidity Multiple interactions between antibody and antigen IgM (with 10 antigen binding sites per molecule) tends to have low affinity but high avidity May be more biologically significant measure than affinity
  • 6. p. 153 Cross-reactivity (p. 141) May contribute to autoimmunity or be protective, depednign on circumstances
  • 7. Precipitation- classic demonstration of antibody-antigen interaction Antibody and soluble antigen aggregate to form a visible precipitate Antibody must be bivalent (Fabs won’t work) Antigen must be multivalent
  • 8. p. 142
  • 9. Precipitation reactions in fluids (p. 142) Precipitation reaction can be seen
  • 10. Precipitation can also be seen in gels Antibody and antigen diffuse toward each other and precipitate where there is equivalence Radial immunodiffusion Double diffusion Immunoelectrophoresis
  • 11. p. 143
  • 12. Agglutination- reaction between antibody and particulate antigen Presence of excess antibody can inhibit agglutination (prozone effect) Each antibody “competes” for epitopes; if it binds one, it cannot link one antigen to another Some antibodies bind but do not agglutinate Epitope density or availability
  • 13. Hemagglutination- red blood cells Well #10- sheep RBC only control 1 through 9- serial twofold dilutions of anti-SRBC p. 145
  • 14. Bacterial agglutination If a patient has a bacterial infection, the patient will produce specific antibodies to that bacterium Serum can be titered with bacterial agglutination reactions Dilutions of serum are tested (usually twofold) example: 1:256 dilution shows agglutination but 1:512 does not Titer is 256
  • 15. Titer can be monitored over time; should decrease as patient recovers Antisera are used to type bacteria, too (against surface antigens, flagellar antigens, etc.) Example: E. coli O157:H7
  • 16. Passive agglutination Soluble antigens don’t agglutinate But if you stick them onto something else (like a latex bead or, historically, a RBC) you can obtain an agglutination reaction Using beads increases sensitivity
  • 17. Agglutination inhibition Current tests are ELISAs p. 146
  • 18. Agglutination inhibition assays Can test for the presence of substances in fluids (e.g., drugs in urine) How? Rubella test is an agglutination inhibition assay; rubella virus causes hemagglutination
  • 19. ELISA- enzyme-linked immunosorbent assay Based on RIA Nearly as sensitive Cheaper and safer Many detection systems have been developed Many variations of the assay have been developed
  • 20. All ELISAs use an antibody conjugated with an enzyme that turns a colorless substrate into a colored product Direct- detects antigens using a single labeled antibody against that antigen Relatively few applications and permutations
  • 21. Indirect ELISA detects antibodies (prior exposure to antigen, e.g., HIV) p. 149 (see also for other types of ELISA designs)
  • 22. Chemiluminescence ELISA technique is the same, detection system is different Luminol, hydrogen peroxide and horseradish peroxidase react to emit light Detection system is more sensitive than the enzyme substrates
  • 23. ELISPOT- can detect secreting cells p. 150
  • 24. The ELISA is very versatile antigen Antibody (mouse Fab, Human Fc) Complement (human) Goat anti-human C1q E Enzyme-conjugated Sheep anti-goat Ab substrate
  • 25. All IgG1 antibodies were identical except for carbohydrate in C H 2
  • 26. Western blotting p. 151
  • 27. Immunofluorescence Antibodies can be labeled with fluorescent dye Can localize binding sites on cells Dyes: Fluorescein, rhodamine, phycoerythrin can be conjugated to Fc region of Ab (so antigen binding is unaffected) Absorb at one wavelength and emit at another
  • 28. Direct and indirect immunofluorescence p. 166
  • 29. p. 166
  • 30. Versatile technique Differentiate T cell subsets Detecting Ag-Ab complexes Localization of target molecules in tissue (variation: immunohistochemical staining)
  • 31. Flow cytometry is quantitative FACS- fluoresence-activated cell sorter Analyze cell populations Sort cells with different features into different containers (e.g., T and B cells; cells that are producing a cell-surface marker from those that are not)
  • 32. p. 155
  • 33. Uses for flow cytometry Percentage of a total population of cells Measuring antigen density within a population of cells Multiple antibodies can be used to assess several cell surface antigens simultaneously Clinical analysis (tumor characterization)
  • 34. Cell culture gene transfection hybridoma technology
  • 35. Summary The structure of antibodies enables them to recognize and bind antigen and to perform appropriate effector functions in response The exquisite specificity and effector activity of antibodies makes them very usefuL in research and diagnostics
  • 36. The organization of immunoglobulin genes allows for the formation of over 10 billion antigen specificities Various in vivo and invitro experimental systems have provided significant insights into the immune response and its regulation