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L2 Practical immunology.pdf

  1. Practical immunology Immunological technologies Dr. B.P. Nayak
  2. Immunological methods: Antibodies as tools •Specific recognition of macromolecules • Cell types: surface molecules (CD markers) • Pathogens • Antibodies •Antibodies as labels •Antibodies as traps •Antibodies as competitors •Assays: • Detection • Localization • Quantitation
  3. A brief history of antibodies • 1891: The term “Antikörper” coined by Paul Ehrlich • Substance in the blood that confers immunity • "if two substances give rise to two different antikörper, then they themselves must be different” • “Lock-and-Key” theory • 1920’s: Heidelberger and Avery identified antibodies as proteins • 1940’s: Linus Pauling confirms lock-and-key theory • 1948:Immunoprecipitation: use of antibodies for detection • 1956: Glick and Chang-Bursa of Fabricius: Antibodies come from B cells • 1976: Hozumi and Tonegawa, antibody gene rearrangement
  4. Useful characteristics of antibodies • Specificity: Ability to recognize individual epitopes • Cross-reactivity: • Ability to bind to more than one epitope • Shared epitopes between different antigens • Strength of binding: • Affinity: Strength of binding between Fab and epitope • Avidity: Overall strength of binding of serum and antigen
  5. Antibody specificity Good: recognize and identify related antigens Bad: Confuse one antigen with a related antigen
  6. Affinity and Avidity Property of the antibody binding site Property of the valency
  7. Antibody types •Polyclonal antibodies: • Isolated from immune serum • Many different idiotypes • Recognize many different epitopes • Strong avidity • Highly sensitive • Cross reactive: Less specific •Monoclonal antibodies: • Single idiotype • Single epitope • Highly specific • Lower avidity
  8. Generation of antibodies as reagents • Polyclonal: • Purify antigen • Inject into rabbit or goat (or other species) • Collect serum • Purify immunoglobulin • Specificity depends on purity of antigen • Monoclonal: • Immunize a mouse • Culture spleen cells fused with myeloma cells • Select clones based on antibody specificity • Specificity to a single epitope
  10. Immunological assays •Diagnostic: •Assay for antigen •Assay for antibody •Research: •Identify cells •Identify cell products •Isolate cells or macromolecules •Assay cell function
  11. Examples Diagnostic assays • Hemaggluination • Hemagglutination inhibition • Enzyme-linked Immunosorbant Assay (ELISA) • Radioimmunoassay (RIA) • Immunofluorescent assay (IFA) Research techniques • ELISA • Immunohistochemistry • Flow cytometry • Cell sorting • ELIspot • Immunoblot • Immunoprecipitation • Mixed lymphocyte response • Chromium release assay
  12. Types of assays •Precipitation tests: Detection based on precipitation of complexes •Colorimetric tests: • Fluorescent • Enzyme-linked
  13. Precipitation reactions: •Antibody-antigen complexes precipitate out at equivalence •Utility: • Detection (visual precipitate) • Quantification • Dilution • Diffusion
  14. “Prozone”
  15. Precipitin tests: Immunodiffusion •Double immunodiffusion: • Well or trough punched in an agarose gel • Antibody and antigen placed in separate well • Precipitate forms if antibody recognizes antigen • Identification of antigen or antigen mix
  16. Radial immunodiffusion • Antibody incorporated into gel • Antigen added to wells • Zone of precipitation is proportional to concentration • Identification and quantification Precipitation at antigen/antibody equivalence
  17. Electrophoresis • Differential charge of antibody and antigen • Improves sensitivity of radial immunodiffusion • Antigen precipitates at equivalence • Height of “rocket” is proportional to antigen concentration Electric current
  18. Quantification by titration •Principle: •Quantitation of antibody or antigen by serial dilution •Single concentration of antigen (or antibody ) •Dilutions of antibody (or antigen) •Titer = the dilution at which precipitation occurs •Reflects but does not equal concentration •Assays: •Hemagglutination •ELISA
  19. Red blood cells: • Easy to see • Bind to antigens Antigen of interest bound to red blood cells Agglutination No agglutination Sample Antibody Titer a 1:32 b Undetectable c 1:16 d 1:128 e 1:256 f Undetectable g 1:8 h 1:32 Prozone
  20. Colorimetric tests: Enzyme-Linked Immunosorbant Assay •ELISA •Principles: • Specific interaction between antibody and antigen • Solid-phase • Sensitive detection •Description: • Antigen bound to a surface • Enzyme-bound antibody • Colorimetric reaction • Semi-quantitative • Generate titer
  21. Variations on ELISA •Indirect ELISA •Capture (Sandwich) ELISA •ELIspot •Competitive ELISA •Radioimmunoassay •Immunoblot (“Western” blot) •Diagnostic kits
  22. Primary goat anti-X Unlabeled Labeled Secondary rabbit anti-goat (labeled)
  23. Competitive ELISA
  24. “Western blot”
  25. Immunochemistry and Immunofluorescence •Solid-phase antigen: • In tissue section • Cytology •Detect presence and distribution of: • Surface marker • Intracellular product • Extracellular product • Metabolic product
  26. Immunofluorescence Direct Indirect
  27. Cell surface markers: Red: Bcl-6, germinal center B cells Green: IRF-4: Plasma cells
  28. B cells: CD45R Plasma cells: IRF-4
  29. Enzyme-linked and related assays Assay Target antigen Detection Variants ELISA Bound to well Chromogen Direct, Indirect, Sandwich ELIspot Product of cultured cells Chromogen B cell or T cell products Immuno- histochemistry In tissue Chromogen, Fluorochrome Direct or indirect Competitive ELISA Soluble Chromogen Immunoblot Gel separated proteins Chromogen, Fluorochrome, Radioactive isotope
  30. Antibodies as traps •Fluorescence-activated cell sorting (FACS): • Quantitation • Isolation •Plate-isolation of cell populations (“panning”) •Magnetic bead isolation of cells and macromolecules
  31. Cells coated with fluorescent antibody B cells: red T cells: green Macrophages: cyan
  32. Lymphocyte assays •Lymphocytes as tools: • Easy to isolate • Survive well in culture •Assays: • ELIspot: B and T helper cell function • Lymphocyte blastogenesis (stimulation) test: T helper cells • Chromium release assay: Cytotoxicity
  33. Magnetic bead isolation Subculture and cloning (blastogenesis)