W7-免疫学常用实验方法.ppt

1、Commonly used immunological methods,Zhihai QinInstitute of BiophysicsChinese Academy of SciencesZ,Ag-Ab reation: ELISA, Immunohistochemistry, FACS, Western blot, Hybridoma & monoclonal antibodyCell function:,proliferation,3HTdR,MTT,CFSE,Cytotoxicity,51Cr,Animal experiment,非共价键结合,I. Antigen-Antibody

2、Reaction,抗体的互补决定区 (CDRs),抗原决定簇(antigenic determinant)又称表位 (epitope),非共价键结合,CDR: Complementarity Determining Region,FR: Framework Region,Complementarity-determining regions (CDRs):,Antigenic Determinant,Specificity 特异性,Features of Ag-Ab Reaction,Reversibility可逆性,Ag+AbAgAb,解离后的抗原或抗体均能保持原有的结构和活性。,在一定条件

3、下(如低pH、高浓度盐等)可以解离。,Ratio最适比例,Sensitivity 敏感性,化学比色法： mg/ml 酶反应测定法： 5-10g/ml (免疫测定中凝胶扩散法和浊度法的敏感度与酶反应法相仿)标记的免疫测定法： ng/ml 水平(例如，用放射免疫测定法或酶免疫测定法测定HBsAg，其敏感度可达0.1ng /ml),ELISA-酶联免疫吸附试验,Immunohistochemistry -免疫组织化学,FACS-流式细胞术,Western Blotting-免疫印迹技术,Often used methods,1. ELISA(酶联免疫吸附试验Enzyme Linked ImmunoS

4、orbent Assay),RIA (radioimmunoassay, Berson&Yalow，1960),IRMA(immunoradiovelric assay, Miles&Hiles,1968),E(L)I(S)A (Engvall&Perlmann, van Weemen and Schuurs , 1971),Copyright 2005 American Association for Clinical Chemistry,Lequin, R. M. Clin Chem 2005;51:2415-2418,Estimates of the number of articles

5、 published per 5-year period from 1960 to 2005,Figure. Estimates of the number of articles published per 5-year period from 1960 to 2005. The search was done in February 2005 in PubMed/National Library of Medicine, NIH, with the search terms: enzyme-immunoassay, enzyme-linked immunosorbent assay (EI

6、A/ELISA combined), and RIA. (Ordinate), number of articles in which the keywords are quoted. (Abscissa), 5-year periods from 1960 to 2005. , combined EIA/ELISA; , RIA. Note: I do not pretend that the numbers in this figure are precise; the trends, however, are evident.,基础: 抗原或抗体的固相化 抗原或抗体的酶标记,用途：目标蛋

7、白的定性或定量分析,三个必要试剂：（1）固相的抗原或抗体， 即免疫吸附剂（immunosorbent）;（2）酶标记的抗原或抗体， 称为结合物（conjugate）；（3）酶反应的底物。,ELISA,a.包被b.抗原抗体反应c.酶促反应,显色d.终止显色，读取数据。,ELISA基本的实验过程,对照和标准曲线,阳性对照阴性对照定量测定：标准品制作标准曲线 待测样品的合理稀释,不同类型的检测方法,2. Immunohistochemistry (免疫组化),原理：抗原抗体反应，抗体标记技术（荧光、酶）用途：组织或细胞内抗原的定性和定位,流程：,Copyright 2007 American Ass

8、ociation for Cancer Research,Zhao, X. et al. Cancer Res 2007;67:4443-4450,Figure 3. mTNF induces infiltration of innate immune cells and angiostasis in tumors from TNFR1/ but not TNFR1/R2/ mice. A to F, TNFR1/ mice or TNFR1/R2/ mice were injected s.c. with 5 x 106 of J-mTNF10 cells. Ten days after t

9、umor cell challenge, tissue sections of the injection site were stained for Mac-1+ (A and B), Gr-1+ (C and D), and CD31+ (E and F) cells as indicated. G and H, for confocal microscopy analysis, tissue sections were stained with Cy3-labeled anti-CD31 for blood vessel endothelial cells (green) and the

10、 VasoTACS in situ kit to detect apoptosis (red). Arrows, apoptotic endothelial cells in the tumor.,成败的关键因素： 组织的固定、包埋 抗体（特异性、浓度、孵育温度和时间） 非特异性抗原的封闭 内源性酶或自发荧光的消减 显色,思考：为什么有的抗体能用于冰冻切片却不能用于石蜡切片？,3. Flow Cytometric analysis(流式细胞术)FACS: (Fluorescence-Activated Cell Sorter),BD FACSVantage,BD-Calibur,测量对象,大小

11、 悬浮在溶液中的相互离散颗粒 大小范围：0.2m - 300 m 高等真核细胞 类型 酵母 细胞类型 细菌 多细胞的聚集体，如胰岛等。 细胞核 非生命颗粒 染色体 其它细胞器以及乳化微球等。,细胞表型分析胞内细胞因子的检测细胞周期和DNA倍体分析细胞分选,主要用途,（1）多参数定量分析每一个细胞； （2）细胞分选； 高纯度：99%以上； 可分析小于1/10000比例的稀有细胞群、 单细胞克隆等。 (3）高通量（分析分选） 分析150，000个/ 秒 分选100，000个/秒,特点,基本结构,工作原理,a. 光信号收集分离，导向，各探测通道接收并转化为电信号。,FSC SSC FL1 FL2 F

12、L3,对数 线性 线性 线性 对数,脉冲处理，模数转换,光信号,电信号,记录数据，显示结果,b. 数据处理,c. 细胞分选,外周全血细胞散射光双参数点图（红细胞溶解后）,流式细胞仪数据分析,非荧光信号,直方图分析,荧光信号,点状图分析,设门分析,可以通过设“门” (Gate)，分析、分选感兴趣的细胞。,实验对照的设计,阴性对照：常用同型抗体对照 阳性对照：单阳性对照（多色分析时用于仪器校正）,抗体的选择,首选直标抗体 荧光分子,实验标本的处理,单细胞悬液的制备 抗体浓度 非特异结合的去除：洗涤和封闭,4. Western Blott,5. Hybridoma Technique & Monoc

13、lonal Antibody Preparation,Jerme，Khler and Milstein 1984 Nobel Prize,B cell,Myeloma,Fusion,B cell,B cell+Bcell,B cell+Myeloma,Myeloma,Myeloma+Myeloma,alive,HGPRT 次嘌呤鸟嘌呤磷酸核糖转移酶(Hypoxanthine guznine phosphoribosyl transferase),TK 胸腺嘧啶激酶 (Thymidine kinase),Preparing mAbs from murine ascites,Pristane (i

14、.p.),Hybridoma (i.p.),ascites,Monoclonal antibody,ELISA,II. Assay for cell function,Proliferation: T cell, tumor cell, Cytotoxic assay : T cell, NK cell, ,Cell Proliferation Stimulators,Nonspecific:Mitogen : LPS, ConA, PHASuperantigen(超抗原): SEBCytokine : IL2, IL4, TNFAntibody: CD3, TCR, CD28 Specifi

15、c:specific antigen:破伤风类毒素，PPD(纯结核蛋白衍生物)MHC: 同种异体细胞(MLR,混合淋巴细胞培养),Assay for T cell proliferation,3H-TdR,wash,Stimulate,Assay,a. 3H-TdR,b. MTT,Assay Mechanism:,操作简单，重复性差,c. CFSE,Figure 15.67 Following cell proliferation in human peripheral blood lymphocytes using the CellTrace CFSE Cell Proliferation

16、Kit C34554). Human peripheral blood lymphocytes were harvested and stained with CellTrace CFSE (carboxyfluorescein diacetate, succinimidyl ester; 5(6)-CFDA, SE) on Day 0. A portion of the population was arrested at the parent generation using mitomycin C (red peak). The remainder of the sample was s

17、timulated with phytohemagglutinin and allowed to proliferate for 5 days. Solid green peaks represent successive generations., for T cell cytotoxicity,Target cell,51Cr labeled,CTL,Target cell,Cr51 release,Assay,CTL,51Cr release,Isolation of stem cells,密度梯度离心法,单抗贴壁铺展法,免疫磁珠分选,流式细胞分选法,III. Technology in

18、volved in stem cell research,Indentification of stem cells,FACS ：造血干细胞可鉴定如下markerCD34，CD33，Sca-1，c-Kit,显微镜技术：生长形态，核型,人类骨髓干细胞,体内分化：胚胎干细胞在裸鼠体内生长分化，形成包含三个胚层来源的良性畸胎瘤,基因芯片：分析基因表达差异,Culture of isolated stem cells,以鼠胚胎干细胞培养为例：,1，饲养细胞的培养2，饲养层的制备,MEF分离,3，小鼠胚胎干细胞分离培养 1）内细胞团的分离与培养 2）ES样细胞扩增 3）单细胞克隆建系 4）鉴定,丝裂霉素

19、C处理或G 射线照射,Differentiation of Stem Cells,悬浮培养法不同的培养基及细胞因子,共培养与不同的支持细胞共培养,III. Animal experiment,Immune deficient mice,裸小鼠(nude mice)，无毛，先天性胸腺发育不良， T细胞功能缺失。,SCID：Severe combined immune deficiency严重联合免疫缺陷小鼠T、B淋巴细胞功能均缺失,A variety of knock-out mice:MT, RAG1-/-, RAG2-/-IFN-/-, IL4R-/- ,Transgenic mice: G

20、FP-transgenic mice,Generation of transgenic mice,Animal experiments,Isolate lymphocytes from spleen or lymph nodes,Isolate tumor cells from tumor tissue.,Adoptive transfer: T, B, sera,Bone Marrow Chimera,Advanced animal experiment,Skin graft,Thank You!,Topics,Immune-mediated angiostasis in tumor dev

21、elopment.MDSC (myeloid-derived suppressor cells) in tumor development.Th17 cells in tumor development. Treg cells in tumor development.Fibroblast in tumor development.The role of TNF and its receptors in tumor immunity.The role of Interferon- and its receptor in tumor immunity.The role of Interleuki

22、n 4 and its receptor in tumor immunity.Cytokines and inflammation resolution.The role of liver fibrosis in development of hepatocellular carcinoma.,要求：每组从topic列表中任选一篇相关文章（Journal of Immunology, Nature, Nature Immunology, The Journal of Clinical Investigation, Cancer Research, Science, The Journal of Experimental Medicine, Nature Medicine; Blood, PNAS, Immunity, Cancer Cell.) Introduction部分要着重阐述，Results部分要说清实验设计思路及过程结果，Discussion部分要指出创新点。,选6名speakers, 2名chairmen20分讲，10分讨论,