w7-免疫學(xué)常用實(shí)驗(yàn)方法

1、Commonly used immunological methods,Zhihai QinInstitute of BiophysicsChinese Academy of SciencesZhihai@ibp.ac.cn,,Ag-Ab reation: ELISA, Immunohistochemistry, FACS, Western blot, Hybridoma & mo

2、noclonal antibodyCell function:,proliferation,,3H－TdR,MTT,CFSE,Cytotoxicity,51Cr,Animal experiment,,非共價(jià)鍵結(jié)合,I. Antigen-Antibody 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,CDR: Complemen

3、tarity Determining Region,FR: Framework Region,Complementarity-determining regions (CDRs):,Antigenic Determinant,Specificity 特異性,Features of Ag-Ab Reaction,Reversibility可逆性,Ag+Ab→Ag·Ab,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

4、(如低pH、高濃度鹽等)可以解離。,Ratio最適比例,Sensitivity 敏感性,化學(xué)比色法： mg/ml 酶反應(yīng)測定法： 5-10μg/ml (免疫測定中凝膠擴(kuò)散法和濁度法的敏感度與酶反應(yīng)法相仿)標(biāo)記的免疫測定法： ng/ml 水平(例如，用放射免疫測定法或酶免疫測定法測定HBsAg，其敏感度可達(dá)0.1ng /ml),ELISA------酶聯(lián)免疫吸附試驗(yàn),Immunohistochemistry --------

5、免疫組織化學(xué),FACS------流式細(xì)胞術(shù),Western Blotting-------免疫印跡技術(shù),Often used methods,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,

6、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 publi

7、shed 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

8、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 2005. 

9、0;, combined EIA/ELISA;  , RIA. [Note: I do not pretend that the numbers in this figure are precise; the trends, however, are evident.],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&quo

10、t;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,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,對照和標(biāo)準(zhǔn)曲線,陽性對照陰性對照定量測定：標(biāo)準(zhǔn)品制作標(biāo)準(zhǔn)曲線 待測樣品的合理稀釋,不同類型的檢測方法,2. Immunohis

11、tochemistry (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：,Copyright ©2007 American Association for Cancer Research,Zhao, X. et al. Cancer Res 2007;67:4443-4450,Figure 3. mTNF induces infiltration of innate i

12、mmune 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

13、 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-CD

14、31 for blood vessel endothelial cells (green) and the VasoTACS in situ kit to detect apoptosis (red). Arrows, apoptotic endothelial cells in the tumor.,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

15、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？,,3. Flow Cytometric analysis(流式細(xì)胞術(shù))FACS: (Fluorescence-Activated Cell Sorter),BD FACSVantage,BD-Calibur,測量對象,大小 懸浮在溶液中的相互離散顆粒 大小范圍：0.2μm - 300 μm

16、 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。,,,細(xì)胞表型分析胞內(nèi)細(xì)胞因子的檢測細(xì)胞周期和DNA倍體分析細(xì)胞分選,主要用途,（1）多參數(shù)定量分析每一個(gè)細(xì)胞；

17、（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）高通量（分析分選）． 分析150，000個(gè)/ 秒 分選100，000個(gè)/秒,,特點(diǎn),基本結(jié)構(gòu),工作原理,a. 光信號收集分離，導(dǎo)向，各探測通道接收并轉(zhuǎn)化為電信號。,,,,,,FSC SSC FL1 FL2 FL3,,,,,,對數(shù)

18、 線性 線性 線性 對數(shù),脈沖處理，模數(shù)轉(zhuǎn)換,光信號,電信號,,,記錄數(shù)據(jù)，顯示結(jié)果,b. 數(shù)據(jù)處理,c. 細(xì)胞分選,外周全血細(xì)胞散射光雙參數(shù)點(diǎn)圖（紅細(xì)胞溶解后）,流式細(xì)胞儀數(shù)據(jù)分析,非熒光信號,直方圖分析,熒光信號,點(diǎn)狀圖分析,設(shè)門分析,可以通過設(shè)“門” (Gate)，分析、分選感興趣的細(xì)胞。,實(shí)驗(yàn)對照的設(shè)計(jì),陰性對照：常用同型抗體對照 陽性對照：單陽性對照（多色分析時(shí)用于儀器校正

19、）,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,4. Western Blott,,5. Hybridoma Technique & Monoclonal Antibody Preparation,Jerme，Köhler and Milstein 1984 Nobel Prize,,B cell,,Myeloma,,,Fusion,,,B ce

20、ll,B cell+Bcell,B cell+Myeloma,Myeloma,Myeloma+Myeloma,,,alive,HGPRT 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),Preparing mAbs from murine ascites,,,,Pristane (i.p.),,,,

21、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, Con

22、A, 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)),Assay for T cell proliferation,3H-TdR,,,,,,,,,,,,,,,,,,,,,,,,,,

23、,,,,,,,,,,,,,,,,,,,,wash,,,,,,,,,,,,,,,,,,,,,,,Stimulate,,Assay,,a. 3H-TdR,b. MTT,Assay Mechanism:,操作簡單，重復(fù)性差,c. CFSE,Figure 15.67 Following cell proliferation in human peripheral blood lymphocytes using the CellTrace CFS

24、E Cell Proliferation 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

25、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 generat

26、ions.,probes.invitrogen.com/handbook/figures/2018.html,,CELL,,,,,,,proliferation,Assay for T cell cytotoxicity,,Target cell,,,51Cr labeled,,,,,,,CTL,,,Target cell,,,,,,Cr51 release,,Assay,,,CTL,51Cr release,Isolation of

27、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,Indentification of stem cells,FACS ：造血干細(xì)胞可鑒定如下marker—CD34，CD33，Sca-1，c-Kit,顯微鏡技術(shù)：生長形態(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)生長分化，形成包含三個(gè)胚層來源的良性畸

28、胎瘤,基因芯片：分析基因表達(dá)差異,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 γ射線照射,Differentiation of Stem Cells,懸浮培養(yǎng)法—不同的培養(yǎng)基及

29、細(xì)胞因子,共培養(yǎng)—與不同的支持細(xì)胞共培養(yǎng),III. Animal experiment,Immune deficient mice,裸小鼠(nude mice)，無毛，先天性胸腺發(fā)育不良， T細(xì)胞功能缺失。,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:μMT, RAG1-/-, RAG2-/-

30、IFNγ-/-, 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, s

31、era…,Bone 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 develop

32、ment. Treg cells in tumor development.Fibroblast in tumor development.The role of TNF and its receptors in tumor immunity.The role of Interferon-γ and its receptor in tumor immunity.The role of Interleukin 4 and its

33、 receptor in tumor immunity.Cytokines and inflammation resolution.The role of liver fibrosis in development of hepatocellular carcinoma.,要求：每組從topic列表中任選一篇相關(guān)文章（Journal of Immunology, Nature, Nature Immunology, The J

34、ournal of Clinical Investigation, Cancer Research, Science, The Journal of Experimental Medicine, Nature Medicine; Blood, PNAS, Immunity, Cancer Cell.) Introduction部分要著重闡述，Results部分要說清實(shí)驗(yàn)設(shè)計(jì)思路及過程結(jié)果，Discussion部分要指出創(chuàng)新點(diǎn)。,選6名