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Immunological techniques

Immunological techniques. Wei Chen, Associate professor Institute of Immunology E-mail:chenwei566@zju.edu.cn http://mypage.zju.edu.cn/566 8888. Objectives.

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Immunological techniques

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  1. Immunological techniques Wei Chen, Associate professor Institute of Immunology E-mail:chenwei566@zju.edu.cn http://mypage.zju.edu.cn/566 8888

  2. Objectives • 􀁺 To know the main laboratory detection method using antibody. • To know how to isolate the specific lymphocyte population. • 􀁺 Be able to describe how to detect the change of human immune function after bacteria or virus infection.

  3. Content • Laboratory methods using antibodies • Lymphocytes isolation • Methods for studying lymphocyte responses • Transgenic mice and gene targeting

  4. Content • Laboratory methods using antibodies • Lymphocytes isolation • Methods for studying lymphocyte responses • Transgenic mice and gene targeting

  5. Laboratory methods using antibodies • Quantitation of Antigen by Immunoassays • Labeling and Detection of Antigens in Cells and Tissues • Measurement of Antigen-Antibody Interactions • Identification and Purification of Proteins

  6. Antigen-Antibody Interactions Agglutination(aggregation) Assays: Immunodiffusion Complement Fixation EIA enzyme immunoassay (IHC/ELISA/ELISPOT) Immunofluorescence (IFA FACS) ICS （Intracellular cytokine staining） CLIA （Chemiluminescence immumoassay） Traditional Immunoassays Modern Immunoassays

  7. 凝集反应 扩散试验 补体结合反应

  8. Immuno-labeling techniques免疫标记技术 • Principle Specific Abs (or Ags ) labelled with fluorescein, enzymes, colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs). b. Types

  9. CD4 CD8 Immuno-labeling techniques horseradish peroxidase IHC Western Blot fluorescence FACS microscope   

  10. Immuno-labeling techniques EIA (Enzyme Immunoassay) Color Detection Substrate

  11. IHC Immunohistochemistry 免疫组织化学

  12. Secondary antibody One conjugated 2nd antibody can detect all primary antibodies with same isotype, which reduce the labor to conjugate all primary antibodies A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications. Specific secondary antibodies are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred.

  13. Secondary antibody

  14. Enzyme immunoassay (EIA)免疫酶测定法 • EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions. • The enzyme converts a colorless substrate (chromogen) to a colored product. • ELISA: Ag or Ab in solution • Enzyme immunohistochemistry: Ag in tissue

  15. ELISA

  16. ELISA

  17. Indirected ELISA

  18. ELISA • BAS（Biotin-avidin system）-ELISA 生物素-亲和素放大系统 一分子亲和素可结合四个分子生物素，敏感、快速，生物素标记抗体、亲和素标记酶。

  19. Biotin-avidin system-ELISA

  20. （Enzyme-linked immuno-sorbent spot，ELISPOT) 灵敏度高，单细胞水平，可高通量筛选

  21. Two cytokines can be detected simultaneously

  22. Immunofluorescence免疫荧光 • Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab. • The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope. • Direct, indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大)

  23. CD4 CD8 Immunofluorescence Immunofluorescence）

  24. Immunofluorescence An Example

  25. Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

  26. Useful tools (1) Usefuldiagram for fluorochomes properties http://www.bdbiosciences.com/research/multicolor/spectrumguide/index.jsp

  27. Useful tools (2) Usefulwebsite tools BD Fluorescence Spectrum Viewer: http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp

  28. Identification and Purification of Proteins Immunoprecipitation. A protein mixture is incubated with specifi c antibody. Any antigen–antibody complexes that form are precipitated from solution by the addition of Protein A-coated beads that bind to the antibodies and collect at the bottom of the tube under the force of centrifugation. After washing, the desired antigen is released from the antibody-bound beads using altered pH and/or high salt concentration

  29. Identification and Purification of Proteins Affinity Chromatography Agarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom. A solution containing antigen is passed slowly through the column, allowing the binding of specifi c antigen to the immobilized antibody. Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing. A solution with the appropriate pH and salt concentration to disrupt Ag–Ab binding is then passed through the column to elute (wash off) the antigen of interest.

  30. Western blotting

  31. Western Blot

  32. Western Blot

  33. Content • Laboratory methods using antibodies • Lymphocytes isolation • Methods for studying lymphocyte responses • Transgenic mice and gene targeting

  34. Isolation of lymphocytes

  35. Magnetic cell sorting (MACS) Three basic steps 1) Target cells are labeled with antibody- conjugated magnetic particles. 2) The labeled cells are placed within a magnetic field. 3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away.

  36. Cell subsets Purification

  37. FACS separation(流式细胞术分离) • The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes. • The modern flow cytometer consists of a light source, collection optics, electronics and a computer to translate signals to data • Isolation of different cell populations by FACS relies on the different expression of surface Ags.

  38. Identification of cell subsets by FACS B cell T cell CD4+ T cell CD8+ Tcells Tregs (CD4+CD25+) Conventional CD4 Immune Cell types and subtypes defined by surface markers (CDs)

  39. Basics of Flow Cytometry • Fluidics • Cells in suspension • flow in single • Optics • scatter light and emit fluorescence • that is collected, filtered • Electronics • converted to digital values • Stored & analyzed on a computer *Collection *Drop charged and collected

  40. FACS Flow cytometry or Fluorescence Activated Cell Sorter (FACS) Quantitative, multi- parameter analysis of large numbers of individual cells Cell surface markers Intracellular proteins Ca++ mobilization 12 colors and 15 parameters Sorting: 70,000 cell/second

  41. FCM

  42. Content • Laboratory methods using antibodies • Lymphocytes isolation • Methods for studying lymphocyte responses • Transgenic mice and gene targeting

  43. T Lymphocyte Function Assays Function Measurement --Proliferation -- CTL killing -- DTH --Apoptosis --Cytokine --HLA detection

  44. T cell proliferation Morphology 3H-thymidine labeling MTT FACS-CFSE staining

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