浙大 高级免疫学技术 英文课件

1、免疫学相关技术 （Technologies in Immunology）,王迪 浙江大学免疫学研究所 wangdi80,Years ago, immunologists typically spent the bulk of their time at the laboratory bench. Their research involved peering into a microscope and probably characterizing the different cells from a blood sample. And their understanding of the i

2、mmune response was limited to what they could see and, based on that, what they could hypothesize.,Before,Now,What is emerging is a more complex understanding of the science that involves many fields of science. Molecular Biology Biochemistry Structural Biology Genomics Proteomics Transgenic and kno

3、ckout animal models,Technology based on Immunology Technology which boost immunology,Antibodies FACS (Analysis and sorting) ELISA ELISPOT Tetramer cell culture (in vitro culture system) Gene Profiling （parallel sequencing） Proteomics Transgenic and knockout animals,Technology based on Immunology Tec

4、hnology which boost immunology,1. Antibodies, Recognise and bind to molecules (antigens) on foreign particles, marking them for destruction. Each antigen may generate several antibodies for different sites (epitopes) on antigen., Proteins secreted by B-lymphocytes (type of white blood cell), in vert

5、ebrates.,170 suppliers in US,Polyclonal antibody,Antigen Immunization,Bleeding,Monoclonal antibody,Freeze,Ab purification *Affinity Purification: Protein A/G, Antigen peptide conjugated on sepharose or agarose beads. Protein A is from the cell wall of S. aureus，MW 42 kDa Protein G is from the cell w

6、all of -hemolytic streptococci, G strain，MW 3035 kDa. Protein A or Protein G can bind to the Fc domain of Ab.,Ab purification,Antibody Purification,Affinity chromatography uses antigen-antibody binding to purify antigens or antibodies To purify a specific antigen from a complex mixture of molecules,

7、 a monoclonal antibody is attached to an insoluble matrix, such as chromatography beads, and the mixture of molecules is passed over the matrix. The specific antibody binds the antigen of interest; other molecules are washed away. Specific antigen is then eluted by altering the pH, which can usually

8、 disrupt antibody-antigen bonds. Antibodies can be purified in the same way on beads coupled to antigen (not shown).,Affinity chromatography,Antibody conjugation,Beads, sepharose Target protein purification Fluorochrome immunohistology, FACS Enzyme Western blot, ELISA Toxin cancer immunotherapy Radi

9、oactive residues Gold Immunoassay,Western Blot fluorescence FACS microscope,Antibody conjugation,Second Antibody,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 cel

10、l sorting applications.,Second Antibody,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.,Tumor Cell,Surface Antigen,Antibody conjugation toxin,Antibody for resea

11、rch unphosphorylated form not recognized,Y,A theoretical experiment comparing Western blot and flow cytometry with three samples and a protein of interest at 1, 10, or 50 copies per cell. Sample 2 and 3 look the same via Western blot, but when stained with fluorescently labeled antibodies, the diffe

12、rences between the samples become more relevant. (Source: P.O. Krutzik et al. / Clinical Immunology 110 (2004) 206221),PhosFLOW和Western blot 的比较,Detection of multi-target cells,CD20 PerCP-Cy5.5,CD3 PE,Zap70 (Y319)/Syk (Y352) Alexa 647,CD3 PE,CD3-/CD20+,CD3+/CD20-,CD3-/CD20-,CD20 PerCP-Cy5.5,Whole Bl

13、ood CD3 CrossLink,CD3/CD28 Crosslink,Genomics & Proteomics - Single Cells Have Big Proteomics Story to Tell.,FACS analysis not only tells you activation of a single cell for one particular phospho protein and pathway, but allows you to study multiple phosphorylation events and pathways simultaneousl

14、y.,一种“三明治”免疫测定法 使用结合有高亲和性抗体的小球，用来特异性地捕获可溶性的抗原. 用荧光检测抗体来检测被捕获的抗原 使用流式细胞仪进行检测分析,Cell lysates: Cytometric Bead Array (CBA),Multiplexed Beads,Shades of a color,Antibody coupled beads, emitting at distinct FL3 intensities,Various analytes,Antibody coupled PE label, emitting at FL2 intensity proportional

15、to analyte conc.,0 pg/mL,80 pg/mL,1250 pg/mL,5000 pg/mL,FL3 Beads,Multiplexed Beads,High throughput - BD CBA,More powerful CBA Flex Set,最多可同时检测72个项目 可自由选择搭配检测项目 检测项目范围更广泛 需要488nm和635nm两种波长的激光器,An Example,Antibodies FACS (Analysis and sorting) ELISA ELISPOT Tetramer cell culture (cells and in vitro c

16、ulture system) Gene Profiling Proteomics Transgenic and knockout animals,Technology based on Immunology Technology which boost immunology,MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells). Biotinylated recombinant class I molecules folded with the

17、peptide of interest and 2M and tetramerized by a fluorescently labeled streptavidin(Streptavidin binds to four biotins per molecule.) . This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex. Antigen specific responses ca

18、n be measured as CD8+, tetramer+ T cells as a fraction of all CD8+ lymphocytes.,Tetramer,Tetramers can bind to three TCRs at once, allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction.,Tetramer,CD8 response related(now CD4 too) and known

19、epitope,Infectious Disease,HIV-AIDS, EBV, CMV, HPV, HBV, HCV, Influenza, Measles, Malaria, TB and RSV,Breast, Prostate, Melanoma, Colon, Lung, Cervical, Ovarian and Leukemia,Diabetes, Lyme disease, Multiple sclerosis, Rheumatoid arthritis, Autoimmune vitiligo,EBV and CMV,Transplantation,Autoimmune D

20、isease,Tumor Immunology,Utility of Tetramer,Antibodies FACS (Analysis and sorting) ELISA ELISPOT Tetramer cell culture (cells and in vitro culture system) Gene Profiling Proteomics Transgenic and knockout animals,Technology based on Immunology Technology which boost immunology,The Biggest cell cultu

21、re resource : American Type Culture Collection (ATCC) Advantage of Cell culture: in vitro study system fast cost less, easy to manipulate Problems in cell culture: 2D vs 3D microenvironment cells mutated during the culture Cell line vs primary cell culture,Cell culture,Primary cell cult

22、ure: growth factors, microenvironment ES cell culture and thymocytes in vitro differentiation: in vivo microenvironment mimic. Specific for lymphoid cells: T and B cells wont grow in vitro, microenvironment determine the function of these cells. Recent advance: stromal cell (mimic microenvironment)

23、In vitro differentiation of thymocytes Adoptive transfer as a cell culture system (in vivo),ES cell culture,ES cell including the newly discovered iPS need to be cultured in cytokine and feeder cells.,In vitro thymocytes differentiation,A small animal model for studying human immune responses to inf

24、ection and cancer,Hematopoietic stem cells,Immunodeficient (scid) mice,NOD-scid,c-/- M. Ito et al., Blood 100:3175 (2002) L. Shultz et al., J. Immunol. 174:6477 (2005) F. Ishikawa et al., Blood 106:1565 (2005) BALB/c-Rag2-/-,c-/- E. Traggiai et al., Science 304:104 (2004),Antibodies FACS (Analysis a

25、nd sorting) ELISA ELISPOT Tetramer cell culture (cells and in vitro culture system) Gene Profiling (massive sequencing) Proteomics Transgenic and knockout animals,Technology based on Immunology Technology which boost immunology,Proteomics: 2-Dimensional Electrophoresis,Antibodies FACS (Analysis and

26、sorting) ELISA ELISPOT Tetramer cell culture (cells and in vitro culture system) Gene Profiling (massive sequencing) Proteomics Transgenic and knockout animals,Technology based on Immunology Technology which boost immunology,Microinjection into the germ line - transgenic animal,Gene injected into th

27、e male pronuclei,Some of the drawbacks of these methods are: The inserted DNA randomly integrates into the genome The eggs must be harvested & fertilized in vitro More than one copy of the gene may get into the genome,Transgenic Animal Generation,Tissue specific promoters,Tissue-specific promoters c

28、an be used to drive the expression of transgene only in desired tissue or cell. For immunologists: Lck promoter Thymocytes and T cells CD19 B lymphocytes CD11c dendritic cells,Mouse “Knock-out” Technology Gene Targeting,Knock-out technology allows for the specific loss of a gene in mice Allows for t

29、he function of the KOd gene to be deduced from the defects seen in the mice can be used to mimick some disease Unlike traditional transgenics the trangene is targeted to a specific site in the DNA of the mouse,target,targeting vector,neor,neor,homologous recombination,“knockout”,gene,microinjection,

30、gene,“transgenic”,TK,Mouse Knock-outs require embryonic stem (ES) cells These are derived from the inner cell mass (ICM) of a blastocyst (the ICM is what will become the fetus) ES cells are pluripotent meaning they can become all the different cell types found in an adult,Positive and Negative Selection of Targeted ES Cells,Recombinants with random insertion,Selection for neor positive,Selection for TK negative,ES cells with homologous recombination,neor,TK,