免疫学常用实验方法.ppt

1、Commonly used immunological methods,Zhihai Qin Institute of Biophysics Chinese Academy of Sciences Z,Ag-Ab reation: ELISA, Immunohistochemistry, FACS, Western blot, Hybridoma 51:2415-2418,Estimates of the number of articles published per 5-year period from 1960 to 2005,Figure. Estimates of the numbe

2、r 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 (EIA/ELISA combined), and RIA. (Ordinate), number of articles in which the keywo

3、rds 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（con

4、jugate）； （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 Association for Cancer Research,Zhao, X. et al. Cancer Res 2007;67:4443

5、-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 tumor cell challenge, tissue sections of the injection site were stai

6、ned 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 VasoTACS in situ kit to detect apoptosis (red). Arrows, apoptotic e

7、ndothelial 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 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。

8、 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 FL3,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

9、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 5(6)-CFDA, SE) on Day 0. A portion of the population was arrested at th

10、e parent generation using mitomycin C (red peak). The remainder of the sample was stimulated with phytohemagglutinin and allowed to proliferate for 5 days. Solid green peaks represent successive generations.,CELL,proliferation,Assay for T cell cytotoxicity,Target cell,51Cr labeled,CTL,Target cell,Cr

11、51 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 involved 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 isolat

12、ed 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 deficien

13、cy 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: GFP-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

14、 Marrow Chimera,Advanced animal experiment,Skin graft,Thank You!,Topics,Immune-mediated angiostasis in tumor development. 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 a

15、nd its receptors in tumor immunity. The role of Interferon- and its receptor in tumor immunity. The role of Interleukin 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 Cl