1、Commonly used immunological methodsZhihai QinInstitute of BiophysicsChinese Academy of SciencesZhihai整理課件Ag-Ab reation: ELISA, Immunohistochemistry, FACS, Western blot, Hybridoma & monoclonal antibodyCell function: proliferation3HTdR MTTCFSECytotoxicity 51CrAnimal experiment整理課件非共價(jià)鍵結(jié)合I. Antigen-Anti

2、body Reaction 抗體的互補(bǔ)決定區(qū) (CDRs)抗原決定簇(antigenic determinant)又稱表位 (epitope)非共價(jià)鍵結(jié)合整理課件CDR1CDR2CDR3CDR1CDR2CDR3CDR: Complementarity Determining RegionFR: Framework Region FR1CDR1FR2CDR2FR3CDR3FR4HLComplementarity-determining regions (CDRs): 整理課件Antigenic Determinant整理課件Specificity 特異性 Features of Ag-Ab Re

3、action 整理課件Reversibility可逆性 Ag+AbAgAb解離后的抗原或抗體均能保持原有的結(jié)構(gòu)和活性。在一定條件下(如低pH、高濃度鹽等)可以解離。整理課件Ratio最適比例整理課件Sensitivity 敏感性化學(xué)比色法： mg/ml 酶反應(yīng)測(cè)定法： 5-10g/ml (免疫測(cè)定中凝膠擴(kuò)散法和濁度法的敏感度與酶反應(yīng)法相仿)標(biāo)記的免疫測(cè)定法： ng/ml 水平(例如，用放射免疫測(cè)定法或酶免疫測(cè)定法測(cè)定HBsAg，其敏感度可達(dá)0.1ng /ml) 整理課件ELISA-酶聯(lián)免疫吸附試驗(yàn)Immunohistochemistry -免疫組織化學(xué)FACS-流式細(xì)胞術(shù)Western Blo

4、tting-免疫印跡技術(shù) Often used methods整理課件1. ELISA(酶聯(lián)免疫吸附試驗(yàn)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)整理課件Copyright 2005 American Association for Clinical ChemistryLequ

5、in, R. M. Clin Chem 2005;51:2415-2418Estimates of the number of articles published per 5-year period from 1960 to 2005Figure. 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

6、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 2005. , combined EIA/ELISA; , RIA. Note: I do not pretend that the numbers in this figure are pr

7、ecise; the trends, however, are evident. 整理課件基礎(chǔ): 抗原或抗體的固相化 抗原或抗體的酶標(biāo)記用途：目標(biāo)蛋白的定性或定量分析 三個(gè)必要試劑：（1）固相的抗原或抗體， 即免疫吸附劑（immunosorbent）;（2）酶標(biāo)記的抗原或抗體， 稱為結(jié)合物（conjugate）；（3）酶反應(yīng)的底物。 ELISA整理課件a.包被b.抗原抗體反應(yīng)c.酶促反應(yīng),顯色d.終止顯色，讀取數(shù)據(jù)。ELISA基本的實(shí)驗(yàn)過程對(duì)照和標(biāo)準(zhǔn)曲線陽(yáng)性對(duì)照陰性對(duì)照定量測(cè)定：標(biāo)準(zhǔn)品制作標(biāo)準(zhǔn)曲線 待測(cè)樣品的合理稀釋整理課件不同類型的檢測(cè)方法整理課件整理課件2. Immunohistochem

8、istry (免疫組化) 原理：抗原抗體反應(yīng)，抗體標(biāo)記技術(shù)（熒光、酶）用途：組織或細(xì)胞內(nèi)抗原的定性和定位流程：整理課件Copyright 2007 American Association for Cancer ResearchZhao, X. et al. Cancer Res 2007;67:4443-4450Figure 3. mTNF induces infiltration of innate immune cells and angiostasis in tumors from TNFR1/ but not TNFR1/R2/ mice. A to F, TNFR1/ mice o

9、r 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 were s

10、tained 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. 整理課件成敗的關(guān)鍵因素： 組織的固定、包埋 抗體（特異性、濃度、孵育溫度和時(shí)間） 非特異性抗原的封閉 內(nèi)源性酶或自發(fā)熒光的消減 顯色思考：為什么有的抗體能用于冰凍切片卻不能用于石蠟切片？整理課件3. Flow Cytometric a

11、nalysis(流式細(xì)胞術(shù))FACS: (Fluorescence-Activated Cell Sorter)BD FACSVantageBD-Calibur 整理課件測(cè)量對(duì)象 大小 懸浮在溶液中的相互離散顆粒 大小范圍：0.2m - 300 m 高等真核細(xì)胞 類型 酵母 細(xì)胞類型 細(xì)菌 多細(xì)胞的聚集體，如胰島等。 細(xì)胞核 非生命顆粒 染色體 其它細(xì)胞器以及乳化微球等。 整理課件細(xì)胞表型分析胞內(nèi)細(xì)胞因子的檢測(cè)細(xì)胞周期和DNA倍體分析細(xì)胞分選主要用途整理課件（1）多參數(shù)定量分析每一個(gè)細(xì)胞； （2）細(xì)胞分選； 高純度：99%以上； 可分析小于1/10000比例的稀有細(xì)胞群、 單細(xì)胞克隆等。 (3

12、）高通量（分析分選） 分析150，000個(gè)/ 秒 分選100，000個(gè)/秒 特點(diǎn)整理課件 基本結(jié)構(gòu)整理課件工作原理整理課件二色鏡 1 2 3帶通濾波片光電倍增管激光束細(xì)胞收集透鏡前向散射光側(cè)向散射光，熒光a. 光信號(hào)收集分離，導(dǎo)向，各探測(cè)通道接收并轉(zhuǎn)化為電信號(hào)。整理課件FSC SSC FL1 FL2 FL3對(duì)數(shù) 線性 線性 線性 對(duì)數(shù)脈沖處理，模數(shù)轉(zhuǎn)換光信號(hào)電信號(hào)記錄數(shù)據(jù)，顯示結(jié)果b. 數(shù)據(jù)處理整理課件c. 細(xì)胞分選488 nm laser+-Charged PlatesSingle cells sortedinto test tubesFALS SensorFluorescence dete

13、ctor整理課件外周全血細(xì)胞散射光雙參數(shù)點(diǎn)圖（紅細(xì)胞溶解后）顆粒度細(xì)胞大小流式細(xì)胞儀數(shù)據(jù)分析非熒光信號(hào)整理課件直方圖分析熒光信號(hào)整理課件點(diǎn)狀圖分析整理課件設(shè)門分析 可以通過設(shè)“門” (Gate)，分析、分選感興趣的細(xì)胞。整理課件 實(shí)驗(yàn)對(duì)照的設(shè)計(jì) 陰性對(duì)照：常用同型抗體對(duì)照 陽(yáng)性對(duì)照：?jiǎn)侮?yáng)性對(duì)照（多色分析時(shí)用于儀器校正） 抗體的選擇 首選直標(biāo)抗體 熒光分子 實(shí)驗(yàn)標(biāo)本的處理 單細(xì)胞懸液的制備 抗體濃度 非特異結(jié)合的去除：洗滌和封閉 整理課件4. Western Blott整理課件5. Hybridoma Technique & Monoclonal Antibody PreparationJerm

14、e，Khler and Milstein 1984 Nobel PrizeB cellMyelomaSecrete AbClonalA clonal cell line secreting specific Ab?整理課件B cellMyelomaFusionB cellB cell+BcellB cell+MyelomaMyelomaMyeloma+Myelomaalive整理課件B cell+MyelomaMyelomaMyeloma+MyelomaDNAA(氨基喋呤)HGPRT+， TK+ H, 次黃嘌呤 T, 胸腺嘧啶HGPRT-， TK-DieHGPRT-， TK- H, 次黃嘌呤

15、T, 胸腺嘧啶A clonal cell line secreting specific Ab-HybridomaHGPRT 次嘌呤鳥嘌呤磷酸核糖轉(zhuǎn)移酶(Hypoxanthine guznine phosphoribosyl transferase) TK 胸腺嘧啶激酶 (Thymidine kinase)整理課件整理課件Preparing mAbs from murine ascitesPristane (i.p.)Hybridoma (i.p.)ascitesMonoclonal antibodyELISA 整理課件II. Assay for cell functionProliferat

16、ion: T cell, tumor cell, Cytotoxic assay : T cell, NK cell, 整理課件Cell Proliferation StimulatorsNonspecific:Mitogen : LPS, ConA, PHASuperantigen(超抗原): SEBCytokine : IL2, IL4, TNFAntibody: CD3, TCR, CD28 Specific:specific antigen:破傷風(fēng)類毒素，PPD(純結(jié)核蛋白衍生物)MHC: 同種異體細(xì)胞(MLR,混合淋巴細(xì)胞培養(yǎng))整理課件 Assay for T cell prolif

17、eration3H-TdR washStimulateAssaya. 3H-TdR整理課件b. MTTAssay Mechanism: 操作簡(jiǎn)單，重復(fù)性差整理課件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 CellTrac

18、e 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 stimulated with phytohemagglutinin and allowed to proliferate for 5 days. Solid green pea

19、ks represent successive generations. /handbook/figures/2018.html CELLproliferation整理課件 Assay for T cell cytotoxicityTarget cell51Cr labeledTarget cellCTLTarget cellCr51 release AssayCTL51Cr release整理課件Isolation of stem cells密度梯度離心法單抗貼壁鋪展法免疫磁珠分選流式細(xì)胞分選法III. Technology involved in stem cell research整理課

20、件Indentification of stem cellsFACS ：造血干細(xì)胞可鑒定如下markerCD34，CD33，Sca-1，c-Kit顯微鏡技術(shù)：生長(zhǎng)形態(tài)，核型人類骨髓干細(xì)胞體內(nèi)分化：胚胎干細(xì)胞在裸鼠體內(nèi)生長(zhǎng)分化，形成包含三個(gè)胚層來(lái)源的良性畸胎瘤基因芯片：分析基因表達(dá)差異整理課件Culture of isolated stem cells以鼠胚胎干細(xì)胞培養(yǎng)為例：1，飼養(yǎng)細(xì)胞的培養(yǎng)2，飼養(yǎng)層的制備MEF分離3，小鼠胚胎干細(xì)胞分離培養(yǎng) 1）內(nèi)細(xì)胞團(tuán)的分離與培養(yǎng) 2）ES樣細(xì)胞擴(kuò)增 3）單細(xì)胞克隆建系 4）鑒定絲裂霉素C處理或G 射線照射整理課件Differentiation of St

21、em Cells懸浮培養(yǎng)法不同的培養(yǎng)基及細(xì)胞因子共培養(yǎng)與不同的支持細(xì)胞共培養(yǎng)整理課件III. Animal experiment Immune deficient mice裸小鼠(nude mice)，無(wú)毛，先天性胸腺發(fā)育不良， T細(xì)胞功能缺失。整理課件SCID：Severe combined immune deficiency嚴(yán)重聯(lián)合免疫缺陷小鼠T、B淋巴細(xì)胞功能均缺失A variety of knock-out mice:MT, RAG1-/-, RAG2-/-IFN-/-, IL4R-/- 整理課件Transgenic mice: GFP-transgenic mice整理課件Gener

22、ation of transgenic mice 整理課件Animal experiments整理課件整理課件整理課件Isolate lymphocytes from spleen or lymph nodes Isolate tumor cells from tumor tissue.整理課件Adoptive transfer: T, B, sera Bone Marrow ChimeraAdvanced animal experimentSkin graft整理課件Thank You!整理課件TopicsImmune-mediated angiostasis in tumor development.MDSC (myeloid-derived suppressor cells