1、Ag-Ab reation:ELISA,Immunohistochemistry,FACS,Western blot,Hybridoma&monoclonal antibodyCell function:proliferation3HTdR MTTCFSECytotoxicity 51CrAnimal experiment非共价键结合I.Antigen-Antibody Reaction 抗体的互补决定区抗体的互补决定区(CDRs)抗原决定簇抗原决定簇(antigenic determinant)又称表位又称表位(epitope)非共价键结合非共价键结合CDR1CDR2CDR3CDR1CDR2
2、CDR3CDR:Complementarity Determining RegionFR:Framework Region FR1CDR1FR2CDR2FR3CDR3FR4HLComplementarity-determining regions(CDRs):Antigenic DeterminantSpecificity 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。解离后的抗原或抗体均能保持原有的结构和活性。在一定条件下在一定条件下(如低如低pH、高浓度盐等、高浓度盐等)可以解离
3、。可以解离。RatioRatio最适比例最适比例Sensitivity Sensitivity 敏感性敏感性化学比色法：化学比色法：mg/ml mg/ml 酶反应测定法：酶反应测定法：5-10g/ml 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)标记的免疫测定法标记的免疫测定法：ng/ml ng/ml 水平水平(例如，用放射免疫测定法或酶免疫测定法测定例如，用放射免疫测定法或酶免疫测定法测定HBsAgHBsAg，其，其敏感度可达敏感度可达0.1ng/ml)0.1ng/ml)ELISA-酶联免疫吸附试验Immunohis
4、tochemistry-免疫组织化学FACS-流式细胞术Western Blotting-免疫印迹技术 Often used methods1.ELISA(酶联免疫吸附试验酶联免疫吸附试验Enzyme Linked ImmunoSorbent 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 2019 American Associa
5、tion for Clinical ChemistryLequin,R.M.Clin Chem 2019;51:2415-2418Estimates of the number of articles published per 5-year period from 1960 to 2019Figure.Estimates of the number of articles published per 5-year period from 1960 to 2019.The search was done in February 2019 in PubMed/National Library o
6、f Medicine,NIH,with the search terms:enzyme-immunoassay,enzyme-linked immunosorbent assay(EIA/ELISA combined),and RIA.(Ordinate),number of articles in which the keywords are quoted.(Abscissa),5-year periods from 1960 to 2019.,combined EIA/ELISA;,RIA.Note:I do not pretend that the numbers in this fig
7、ure are precise;the trends,however,are evident.基础基础:抗原或抗体的固相化 抗原或抗体的酶标记用途：用途：目标蛋白的定性或定量分析 三个必要试剂三个必要试剂：（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。ELISAa.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。ELISAELISA基本的实验过程基本的实验过程对照和标准曲线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不同类型的检
8、测方法2.Immunohistochemistry(免疫组化免疫组化)原理：原理：抗原抗体反应，抗体标记技术（荧光、酶）用途：用途：组织或细胞内抗原的定性和定位流程：流程：Copyright 2019 American Association for Cancer ResearchZhao,X.et al.Cancer Res 2019;67:4443-4450Figure 3.mTNF induces infiltration of innate immune cells and angiostasis in tumors from TNFR1/but not TNFR1/R2/mice.A
9、 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 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 w
10、ere stained with Cy3-labeled anti-CD31 for blood vessel endothelial cells(green)and the VasoTACS in situ kit to detect apoptosis(red).Arrows,apoptotic endothelial cells in the tumor.成败的关键因素成败的关键因素：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？思考：为什么有的抗体能用于冰冻切片却不能用于石蜡
11、切片？3.Flow Cytometric analysis(流式细胞术流式细胞术)FACS:(Fluorescence-Activated Cell Sorter)BD FACSVantageBD-Calibur 测量对象测量对象 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。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
12、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 特点特点 基本结构基本结构工作原理工作原理二色镜 1 2 3带通滤波片光电倍增管激光束细胞收集透镜前向散射光侧向散射光，荧光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对数对数 线性线性 线性线性 线性线性 对数对数脉冲处理，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488 nm laser+-Charged PlatesSingle cells sortedinto test tubesFALS SensorFluoresce
13、nce detector外周全血细胞散射光双参数点图（红细胞溶解后）颗粒度细胞大小流式细胞仪数据分析流式细胞仪数据分析非荧光信号非荧光信号直方图分析直方图分析荧光信号荧光信号点状图分析点状图分析设门分析设门分析 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）抗体的选择抗体的选择 首选直标抗体 荧光分子 实验标本的处理实验标本的处理 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 Blott5.Hybridoma Technique&Monoclonal
14、Antibody PreparationJerme，Khler and Milstein 1984 Nobel PrizeB cellMyelomaSecrete AbClonalA clonal cell line secreting specific Ab?B cellMyelomaFusionB cellB cell+Bcell B cell+MyelomaMyelomaMyeloma+MyelomaaliveB cell+MyelomaMyelomaMyeloma+MyelomaDNAA(氨基喋呤氨基喋呤)H,次黄嘌呤次黄嘌呤 T,胸腺嘧啶胸腺嘧啶Die H,次黄嘌呤次黄嘌呤 T,胸腺
15、嘧啶胸腺嘧啶A clonal cell line secreting specific Ab-HybridomaHGPRT 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 ascitesPristane(i.p.)Hybridoma(i.p.)ascitesMonoclonal antibodyELISA II.Assay for cell functionProliferation:T cell,tumor c
16、ell,Cytotoxic assay:T cell,NK cell,Cell Proliferation StimulatorsNonspecific:Mitogen:LPS,ConA,PHASuperantigen(超抗原超抗原):SEBCytokine:IL2,IL4,TNFAntibody:CD3,TCR,CD28 Specific:specific antigen:破伤风类毒素，破伤风类毒素，PPD(纯结核蛋白衍生物纯结核蛋白衍生物)MHC:同种异体细胞同种异体细胞(MLR,混合淋巴细胞培养混合淋巴细胞培养)Assay for T cell proliferation3H-TdR w
17、ashStimulateAssaya.3H-TdRb.MTTAssay Mechanism:操作简单，重复性差c.CFSEFigure 15.67 Following cell proliferation in human peripheral blood lymphocytes using the CellTrace CFSE Cell Proliferation Kit C34554).Human peripheral blood lymphocytes were harvested and stained with CellTrace CFSE(carboxyfluorescein di
18、acetate,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 stimulated with phytohemagglutinin and allowed to proliferate for 5 days.Solid green peaks represent successive generations.p
19、robes.invitrogen/handbook/figures/2018.html CELLproliferation Assay for T cell cytotoxicityTarget cell51Cr labeledTarget cellCTLTarget cellCr51 release AssayCTL51Cr releaseIsolation of stem cells密度梯度离心法密度梯度离心法单抗贴壁铺展法单抗贴壁铺展法免疫磁珠分选免疫磁珠分选流式细胞分选法流式细胞分选法III.Technology involved in stem cell researchIndent
20、ification of stem cellsFACS：造血干细胞可鉴定如下markerCD34，CD33，Sca-1，c-Kit显微镜技术：显微镜技术：生长形态，核型人类骨髓干细胞体内分化：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：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：以鼠胚胎干细胞培养为例：1，饲养细胞的培养，饲养细胞的培养2，饲养层的制备，饲养层的制备MEF分离分离3，小鼠胚胎干细胞分离培养，小鼠胚胎干细胞分离培养 1）内细胞团的分离与培养）内细胞团的分离与培养 2）ES样细胞
21、扩增样细胞扩增 3）单细胞克隆建系）单细胞克隆建系 4）鉴定）鉴定丝裂霉素丝裂霉素C处理或处理或G 射线照射射线照射Differentiation of Stem Cells悬浮培养法悬浮培养法不同的培养基及细胞因子不同的培养基及细胞因子共培养共培养与不同的支持细胞共培养与不同的支持细胞共培养III.Animal experiment Immune deficient mice裸小鼠裸小鼠(nude mice)，无毛，无毛，先天性胸腺发育不良，先天性胸腺发育不良，T细细胞功能缺失。胞功能缺失。SCID：Severe combined immune deficiency严重联合免疫缺陷小鼠严重联
22、合免疫缺陷小鼠T、B淋巴细胞功能均缺失淋巴细胞功能均缺失A variety of knock-out mice:MT,RAG1-/-,RAG2-/-IFN-/-,IL4R-/-Transgenic mice:GFP-transgenic miceGeneration of transgenic mice Animal experimentsIsolate lymphocytes from spleen or lymph nodes Isolate tumor cells from tumor tissue.Adoptive transfer:T,B,sera Bone Marrow Chime
23、raAdvanced animal experimentSkin graftThank You!TopicsImmune-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 and its receptors in tumor
24、 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：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。选选6名名speakers,2名名chairmen 20分讲，10分讨论
