免疫学常用实验方法.ppt

Commonlyusedimmunologicalmethods,ZhihaiQinInstituteofBiophysicsChineseAcademyofSciencesZhihaiibp.ac,Ag-Abreation:ELISA,Immunohistochemistry,FACS,Westernblot,Hybridoma51:2415-2418,Estimatesofthenumberofarticlespublishedper5-yearperiodfrom1960to2019,Figure.Estimatesofthenumberofarticlespublishedper5-yearperiodfrom1960to2019.ThesearchwasdoneinFebruary2019inPubMed/NationalLibraryofMedicine,NIH,withthesearchterms:enzyme-immunoassay,enzyme-linkedimmunosorbentassay(EIA/ELISAcombined),andRIA.(Ordinate),numberofarticlesinwhichthekeywordsarequoted.(Abscissa),5-yearperiodsfrom1960to2019.,combinedEIA/ELISA;,RIA.Note:Idonotpretendthatthenumbersinthisfigureareprecise;thetrends,however,areevident.,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（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：,Copyright2019AmericanAssociationforCancerResearch,Zhao,X.etal.CancerRes2019;67:4443-4450,Figure3.mTNFinducesinfiltrationofinnateimmunecellsandangiostasisintumorsfromTNFR1/butnotTNFR1/R2/mice.AtoF,TNFR1/miceorTNFR1/R2/micewereinjecteds.c.with5x106ofJ-mTNF10cells.Tendaysaftertumorcellchallenge,tissuesectionsoftheinjectionsitewerestainedforMac-1+(AandB),Gr-1+(CandD),andCD31+(EandF)cellsasindicated.GandH,forconfocalmicroscopyanalysis,tissuesectionswerestainedwithCy3-labeledanti-CD31forbloodvesselendothelialcells(green)andtheVasoTACSinsitukittodetectapoptosis(red).Arrows,apoptoticendothelialcellsinthetumor.,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.FlowCytometricanalysis(流式细胞术)FACS:(Fluorescence-ActivatedCellSorter),BDFACSVantage,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-300m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。,FSCSSCFL1FL2FL3,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.WesternBlott,5.HybridomaTechnique5(6)-CFDA,SE)onDay0.AportionofthepopulationwasarrestedattheparentgenerationusingmitomycinC(redpeak).Theremainderofthesamplewasstimulatedwithphytohemagglutininandallowedtoproliferatefor5days.Solidgreenpeaksrepresentsuccessivegenerations.,probes.invitrogen/handbook/figures/2018.html,CELL,proliferation,AssayforTcellcytotoxicity,Targetcell,51Crlabeled,CTL,Targetcell,Cr51release,Assay,CTL,51Crrelease,Isolationofstemcells,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.Technologyinvolvedinstemcellresearch,Indentificationofstemcells,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,Cultureofisolatedstemcells,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,DifferentiationofStemCells,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.Animalexperiment,Immunedeficientmice,裸小鼠(nudemice)，无毛，先天性胸腺发育不良，T细胞功能缺失。,SCID：Severecombinedimmunedeficiency严重联合免疫缺陷小鼠T、B淋巴细胞功能均缺失,Avarietyofknock-outmice:MT,RAG1-/-,RAG2-/-IFN-/-,IL4R-/-,Transgenicmice:GFP-transgenicmice,Generationoftransgenicmice,Animalexperiments,Isolatelymphocytesfromspleenorlymphnodes,Isolatetumorcellsfromtumortissue.,Adoptivetransfer:T,B,sera,BoneMarrowChimera,Advancedanimalexperiment,Skingraft,ThankYou!,Topics,Immune-mediatedangiostasisintumordevelopment.MDSC(myeloid-derivedsuppressorcells)intumordevelopment.Th17cellsintumordevelopment.Tregcellsintumordevelopment.Fibroblastintumordevelopment.TheroleofTNFanditsreceptorsintumorimmunity.TheroleofInterferon-anditsreceptorintumorimmunity.TheroleofInterleukin4anditsreceptorintumorimmunity.Cytokinesandinflammationresolution.Theroleofliverfibrosisindevelopmentofhepatocellularcarcinoma.,要求：每组从topic列表中任选一篇相关文章（JournalofImmunology,Nature,NatureImmunology,TheJournalofClinic