免疫學(xué)常用實(shí)驗(yàn)方法

1、commonly used immunological methodszhihai qininstitute of biophysicschinese academy of scienceszag-ab reation: elisa, immunohistochemistry, facs, western blot, hybridoma & monoclonal antibodycell function: proliferation3htdr mttcfsecytotoxicity 51cranimal experiment非共價(jià)鍵結(jié)合i. antigen-antibody reaction

2、 抗體的互補(bǔ)決定區(qū)抗體的互補(bǔ)決定區(qū) (cdrs)抗原決定簇抗原決定簇(antigenic determinant)又稱表位又稱表位 (epitope)非共價(jià)鍵結(jié)合非共價(jià)鍵結(jié)合cdr1cdr2cdr3cdr1cdr2cdr3cdr: complementarity determining regionfr: framework region fr1cdr1fr2cdr2fr3cdr3fr4hlcomplementarity-determining regions (cdrs): antigenic determinantspecificity 特異性 features of ag-ab reac

3、tion 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。解離后的抗原或抗體均能保持原有的結(jié)構(gòu)和活性。在一定條件下在一定條件下(如低如低ph、高濃度鹽等、高濃度鹽等)可以解離?？梢越怆x。ratioratio最適比例最適比例sensitivity sensitivity 敏感性敏感性化學(xué)比色法：化學(xué)比色法： mg/ml mg/ml 酶反應(yīng)測(cè)定法：酶反應(yīng)測(cè)定法： 5-10g/ml 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)定法標(biāo)記的免疫測(cè)定法： ng/

4、ml ng/ml 水平水平( (例如，用放射免疫測(cè)定法或酶免疫測(cè)定法測(cè)定例如，用放射免疫測(cè)定法或酶免疫測(cè)定法測(cè)定hbsaghbsag，其，其敏感度可達(dá)敏感度可達(dá)0.1ng /ml) 0.1ng /ml) 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 blotting-免疫印跡技術(shù) often used methods1. elisa(酶聯(lián)免疫吸附試驗(yàn)酶聯(lián)免疫吸附試驗(yàn)enzyme linked immunosorbent assay)ria (radioimmunoassay, berson&yalow，1960) irm

5、a(immunoradiovelric assay, miles&hiles,1968)e(l)i(s)a (engvall&perlmann, van weemen and schuurs , 1971)copyright 2005 american association for clinical chemistrylequin, r. m. clin chem 2005;51:2415-2418estimates of the number of articles published per 5-year period from 1960 to 2005figure. estimates

6、 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 search terms: enzyme-immunoassay, enzyme-linked immunosorbent assay (eia/elisa combined), and ria. (ordinate), number of articles in wh

7、ich 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 precise; the trends, however, are evident. 基礎(chǔ)基礎(chǔ): 抗原或抗體的固相化 抗原或抗體的酶標(biāo)記用途：用途：目標(biāo)蛋白的定性或定量分析 三個(gè)必要試劑三個(gè)必要試劑：（1）固相的抗原或抗體， 即免疫吸附劑（immunosorbent）;（2

8、）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。 elisaa.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)。elisaelisa基本的實(shí)驗(yàn)過(guò)程基本的實(shí)驗(yàn)過(guò)程對(duì)照和標(biāo)準(zhǔn)曲線對(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)樣品的合理稀釋不同類型的檢測(cè)方法不同類型的檢測(cè)方法2. immunohistochemistry (免疫組化免疫組化) 原理：原理：抗原抗體反應(yīng)，抗體標(biāo)記技術(shù)（熒光、酶）用途：用途：組織或細(xì)胞內(nèi)抗原的定性和定位流程：流程：copyright 2007 american association for cancer r

9、esearchzhao, 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 or tnfr1/r2/ mice were injected s.c. with 5 x 106 of j-mtnf10 cells. ten days after tumor cell challenge, ti

10、ssue 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-cd31 for blood vessel endothelial cells (green) and the vasotacs in situ kit t

11、o detect apoptosis (red). arrows, apoptotic endothelial cells in the tumor. 成敗的關(guān)鍵因素成敗的關(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？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)流式細(xì)胞術(shù))facs: (fluorescence-activated cell sorter)bd facsvantagebd-cal

12、ibur 測(cè)量對(duì)象測(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。 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)胞分選主要用途主要用途（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）高通量（分析分選） 分析150，000個(gè)/ 秒 分選100，000個(gè)/秒 特點(diǎn)特點(diǎn) 基本結(jié)構(gòu)基本結(jié)構(gòu)工作原理工作原理二色鏡 1 2 3帶通濾波片光電倍增管激光

13、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)。fsc ssc fl1 fl2 fl3對(duì)數(shù)對(duì)數(shù) 線性線性 線性線性 線性線性 對(duì)數(shù)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c. 細(xì)胞分選488 nm laser+-charged platessingle cells sortedinto test tubesfals sensorfluorescence detector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)分析流式細(xì)胞儀數(shù)據(jù)分析非熒光信號(hào)非熒光信號(hào)直方圖分析直方圖分析熒光信號(hào)熒光信號(hào)點(diǎn)

14、狀圖分析點(diǎn)狀圖分析設(shè)門分析設(shè)門分析 可以通過(guò)設(shè)“門” (gate)，分析、分選感興趣的細(xì)胞。 實(shí)驗(yàn)對(duì)照的設(shè)計(jì)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 blott5. hybridoma technique & monoclonal antibody preparationjerme，khler and milstein 1984 nobel prizeb cellmyelomasecrete

15、 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, 次黃嘌呤次黃嘌呤 t, 胸腺嘧啶胸腺嘧啶die h, 次黃嘌呤次黃嘌呤 t, 胸腺嘧啶胸腺嘧啶a clonal cell line secreting specific ab-hybridomahgprt 次嘌呤鳥(niǎo)嘌呤磷酸核糖轉(zhuǎn)移酶(

16、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 cell, cytotoxic assay : t cell, nk cell, cell proliferation stimulator

17、snonspecific:mitogen : lps, cona, phasuperantigen(超抗原超抗原): sebcytokine : il2, il4, tnfantibody: cd3, tcr, cd28 specific:specific antigen:破傷風(fēng)類毒素，破傷風(fēng)類毒素，ppd(純結(jié)核蛋白衍生物純結(jié)核蛋白衍生物)mhc: 同種異體細(xì)胞同種異體細(xì)胞(mlr,混合淋巴細(xì)胞培養(yǎng)混合淋巴細(xì)胞培養(yǎng)) assay for t cell proliferation3h-tdr washstimulateassaya. 3h-tdrb. mttassay mechanism: 操

18、作簡(jiǎn)單，重復(fù)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 diacetate, succinimidyl ester; 5(6)-cfda, se)

19、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. cellproliferation assay for t cell c

20、ytotoxicitytarget cell51cr labeledtarget cellctltarget cellcr51 release assayctl51cr releaseisolation of stem cells密度梯度離心法密度梯度離心法單抗貼壁鋪展法單抗貼壁鋪展法免疫磁珠分選免疫磁珠分選流式細(xì)胞分選法流式細(xì)胞分選法iii. technology involved in stem cell researchindentification 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)：顯微鏡技術(shù)：生

21、長(zhǎng)形態(tài)，核型人類骨髓干細(xì)胞體內(nèi)分化：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：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：以鼠胚胎干細(xì)胞培養(yǎng)為例：1，飼養(yǎng)細(xì)胞的培養(yǎng)，飼養(yǎng)細(xì)胞的培養(yǎng)2，飼養(yǎng)層的制備，飼養(yǎng)層的制備mef分離分離3，小鼠胚胎干細(xì)胞分離培養(yǎng)，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)）內(nèi)細(xì)胞團(tuán)的分離與培養(yǎng) 2）es樣細(xì)胞擴(kuò)增樣細(xì)胞擴(kuò)增 3）單細(xì)胞克隆建系）單細(xì)胞克隆建系 4）鑒定）鑒定絲裂霉素絲裂霉素c處理或處理或g 射線照射射線照射differentiation of s

22、tem cells懸浮培養(yǎng)法懸浮培養(yǎng)法不同的培養(yǎng)基及細(xì)胞因子不同的培養(yǎng)基及細(xì)胞因子共培養(yǎng)共培養(yǎng)與不同的支持細(xì)胞共培養(yǎng)與不同的支持細(xì)胞共培養(yǎng)iii. animal experiment immune deficient mice裸小鼠裸小鼠(nude mice)，無(wú)毛，無(wú)毛，先天性胸腺發(fā)育不良，先天性胸腺發(fā)育不良， t細(xì)細(xì)胞功能缺失。胞功能缺失。scid：severe combined immune deficiency嚴(yán)重聯(lián)合免疫缺陷小鼠嚴(yán)重聯(lián)合免疫缺陷小鼠t、b淋巴細(xì)胞功能均缺失淋巴細(xì)胞功能均缺失a variety of knock-out mice:mt, rag1-/-, rag2-/-

23、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 chimeraadvanced animal experimentskin graftthank you!topicsimmune-mediated angiostasis in tumor development.mds