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1、免疫新技术在科研中的应用,厦门大学医学院 高丰光,抗原或抗体的检测,抗原抗体反应的特点抗原抗体反应的影响因素常用的抗原抗体反应,抗原抗体反应的特点,高度的特异性可逆性抗原抗体结合除了空间构象互补外，主要以氢键、静电引力、范德华力和疏水键等分子表面的化学基团之间的非共价方式结合。这种非共价键不如共价键结合稳定，极易受温度、酸碱度和离子强度的影响而解离。抗原抗体比例影响免疫复合物的大小抗原抗体反应的两个阶段,抗原抗体反应的影响因素,抗原抗体浓度与比例电解质抗原抗体特异性结合后，亲水性降低，易受电解质的影响而使表面失去较多的负电荷。温度适当的温度可增加抗原与抗体分子的碰撞机会，加速抗原抗体复合物的形
2、成。酸碱度抗原抗体反应的最适pH在68之间，pH过高或过低，即过碱或过酸，均可影响抗原、抗体的理化性质。,常用的抗原抗体反应,凝集反应沉淀反应免疫标记技术,凝集反应,直接凝集反应血型鉴定肥大反应间接凝集反应间接凝集抑制试验微粒捕获酶免疫分析技术,间接凝集反应,将可溶性抗原或抗体吸附在与免疫无关的颗粒载体上，形成致敏颗粒，再与相应抗体或抗原进行反应产生的凝集反应，称为间接凝集反应。颗粒载体有红细胞、聚苯乙烯乳胶颗粒、活性炭颗粒，而相应的凝集现象分别称为间接血球凝集、间接乳胶凝集、间接炭粒凝集反应。,微粒捕获酶免疫分析技术,将已知特异性抗体致敏的免疫微粒与生物素亲和素酶放大系统相结合，最后酶作用于
3、荧光底物，使之发荧光，通过检测荧光强度判断待测抗原的含量。检测肿瘤标记物CAl99、CEA、CAl25、AFP、性激素、甲状腺素T-T+、叶酸、HIV抗体、HCG等微量可溶性抗原。,沉淀反应,速率散射比浊法免疫比浊法琼脂扩散法单向琼脂扩散双向琼脂扩散火箭电泳对流免疫电泳免疫印迹技术,速率散射比浊法免疫比浊法,将已知抗体与相应抗原在液相中按一定比例混合形成可溶性免疫复合物，这些复合物微小粒子对一定波长的光照射发生散射，可通过三个光路系统测定光散射(OD值)。抗体含量固定并处于抗体过剩时，免疫复合物的多少直接取决于抗原的浓度，抗原的终浓度通过标准品绘出的标准曲线查出。提高了灵敏度，通过自动化，可同
4、时检测多个样品并进行精确的定量分析。检测前白蛋白、酸性蛋白酶、巨球蛋白、转铁蛋白、尿微量蛋白及IgG、IgM、IgA及补体，药物含量分析。,免疫印迹技术,将用SDS聚丙烯酰胺凝胶电泳PAGE分离得到的按分子量大小排列的非标记蛋白转移到固相载体膜上，再用标记的特异性的抗血清或单克隆抗体对蛋白质进行定性及定量分析的技术，其鉴定蛋白质的敏感性为15ng。检测可溶性抗原、细胞成分的鉴定与分析，检测与自身变性细胞核成分结合的抗体(抗核抗体)，HIV的明确诊断。,免疫印迹法的基本步骤,电泳分离蛋白抗原，SDS是一种阴离子去污剂，与蛋白质牢固结合，十二烷基磺酸根带负电荷，使样品中各种蛋白质与SDS形成SDS
5、-蛋白质复合物，由于蛋白质表面均带有相同密度的负电荷，使分子构型几乎相同(线性)。因此SDS-PAGE中，SDS多肽复合物的电泳迁移率只与其分子质量有关，而不受所带电荷及分子形态的影响。将SDS-PAGE分离到的蛋白条带转移至固相的硝酸纤维素膜上。蛋白条带可用酶、同位素标记的一抗或二抗进行特异性反应，加入显色底物或放射自显影以显示结果。,免疫标记技术,免疫酶测定法免疫荧光技术放射免疫测定法免疫胶体金技术,免疫酶测定法,是一种用酶标记一抗或二抗检测特异性抗原或抗体的方法。将抗原抗体反应的高度特异性与酶对底物的高效催化作用有效地结合起来，通过酶分解底物产生有色物质(也可作用于荧光底物，产生荧光)，
6、肉眼观察颜色深浅或酶标仪测定光密度值(OD)，以反映抗原或抗体的含量。本法灵敏度高，检测可溶性抗原或抗体、组织或细胞表面特异性抗原。,ELISA,双抗体夹心法(sandwich assay)检测血清、脑脊液、胸、腹水等各种液相中的可溶性抗原间接法测定细胞及组织表面抗原,酶联免疫斑点试验(enzymelinked immunospot assay，ELISPOT),用已知细胞因子的抗体包被固相载体，加入待检效应细胞，温育一定时间后洗去细胞，如待检效应细胞产生相应细胞因子，则与已包被的抗体结合，再加入酶标记抗该细胞因子抗体，加底物显色。一般选择硝酸纤维素膜(NC)或聚偏二氟乙烯(PVDF)膜覆盖微
7、量反应板作为固相，在分泌相应细胞因子的细胞所在局部呈现有色斑点，一个斑点表示一个分泌相应细胞因子的细胞，通过计数可推算出分泌某种细胞因子细胞的频率。,免疫荧光技术,用荧光素标记一抗或二抗，检测特异性抗原或抗体的方法。常用的荧光素有异硫氰酸荧光素(nuorescein isothiocyanate，FITC)、藻红蛋白(phycoerythrin，PE)等。在激发光的作用下，可直接发射荧光，前者发黄绿色荧光，后者发红色荧光。,Immunofluorescent stain of immunoglobulin G(IgG)showing linear pattern in Goodpastures
8、 syndrome,天疱疮免疫荧光染色,放射免疫测定法,用放射性同位素标记抗原或抗体进行的免疫测定。既有同位素的敏感性又有抗原抗体结合的特异性，同时具有重复性好、准确性高、标本用量少等优点。广泛应用于激素、药物等微量物质的检测。,免疫胶体金技术,氯金酸(HAuCl+)在还原剂作用下，产生分散状态的胶体金颗粒。碱性条件下，金颗粒表面带负电荷，与蛋白质正电荷基团结合。胶体金可标记白蛋白、免疫球蛋白、糖蛋白、激素、脂蛋白、植物血凝素、卵白素等。大分子以单层形式吸附在金颗粒表面。不同还原剂作用于氯金酸，产生的胶体金粒径大小不相同(550nm)，小粒径的胶体金由于穿透性好，电子密度高，常被用于免疫电镜技
9、术。这些小粒径的金颗粒，经银显影液处理后，金粒子还原银离子生成银颗粒而吸附在金颗粒周围呈黑褐色，从而放大了金颗粒的显色效果，又称免疫金银法。胶体金颜色随颗粒大小而变化，大于20 nm的金颗粒在光镜下呈现砖红色，可在光镜水平行免疫分析，也可用银显影剂增强，进一步提高灵敏度。当胶体金的粒径较大、浓度密集时肉眼水平即可观察，即胶体金斑点渗滤试验和胶体金斑点免疫层析试验。,免疫细胞的检测,免疫细胞的分离磁珠分离法fluorescence-activated cell sorter，FACS免疫细胞功能的测定T细胞B细胞,磁珠分离法,特异性分离所需淋巴细胞的方法。将特异性抗体(如抗CD3、抗CD4、抗C
10、D8等)吸附在铁颗粒(磁珠)上，加至细胞悬液中，具有相应抗原的细胞与磁珠上的特异性抗体结合。反应管置于磁场中，铁颗粒受磁场的吸引，携带有相应细胞的磁球吸附于靠近磁铁的管壁上。弃细胞悬液，重新解离细胞与磁珠。,fluorescence-activated cell sorter，FACS,T细胞鉴定及功能测定,使用酶、免疫荧光标记单抗进行鉴定淋巴细胞转化试验E花环形成试验混合淋巴细胞培养CTL介导的细胞毒试验,抗原肽MHC分子四聚体技术tetramer,用生物素化的抗原肽MHC分子复合物与荧光标记的亲合素结合，由于1个荧光素标记的亲合素可结合4个生物素分子，能使4个MHC抗原肽复合物形成一个复合
11、体，将该复合体标记荧光素后，即成抗原特异性四聚体。抗原特异性四聚体能与样品中的特异性T细胞的TCR结合，由于四聚体能同时结合一个T细胞表面的4个TCR，亲和力大大提高。用流式细胞术即可确定待检标本中抗原特异性CTL细胞的频率。MHC分子可为工类或类分子，与抗原肽形成的四聚体复合物，可分别鉴定表达特异性TCR的CD8+T细胞及CD4+T细胞的频率。,B细胞的鉴定及功能测定,检测B细胞分化抗原测定B细胞产生抗体的能力溶血空斑试验ELISPOT,细胞因子的检测,生物活性检测细胞增生或增生抑制法细胞病变抑制法趋化作用测定法免疫学检测法分子生物学技术,基因敲除技术和转基因动物,应用基因同源重组，将外源有
12、功能基因(基因组中原先不存在、或已失活的基因)，转入细胞与基因组中的同源序列进行同源重组，插人到基因组中，在细胞内获得表达。通过同源重组产生目标基因缺失或失活的转基因动物可以确定被敲除的基因在体内代谢过程中的作用，还可确定被敲除基因在分化、发育、生存等过程中的作用和必要性。转基因动物可以作为疾病模型。可以用于药物筛选的动物模型。转基因动物可作为“生物反应器”生产药物。,Th细胞在mCTL介导的肿瘤保护中作用的研究,相关说明,OT-1 mice:表达SIINFEKL特异性TCROVA：鸡卵白蛋白OVA CTL epitope:SIINFEKLOVA Th epitope:ISQAVHAAHAEI
13、-NEAGROVA（OVT）RAG-1 KO mice:C57BL/6 mice:IFA/CFA/KLHEG7 cell lineEL4 cell line,相关步骤,mCTL and eCTL generationOT-1细胞转输RAG-1-基因敲除小鼠，2天后以 OT-1 TCR 特异性表位多肽 SIINFEKL 免疫；specific Th generationOVA 特异性和非特异性Th表位多肽免疫C57BL/6小鼠以产生特异性和非特异性Th细胞；mCTL transfer记忆性CTL转输已产生特异性和非特异性Th的 C57BL/6 小鼠；tumor challenge接种表达OVA抗
14、原的肿瘤细胞EG7；,Generation of Ovalbumin-specific Memory CD8 T Cells.,OT-1 lymph nodes cells were transferred to RAG-/-mice through tail vein injection,and mice were immunized with 50ug of SIINFEKL peptide in CFA 1 day later.At day 7,14,21,mice were sacrificed,and splenocytes or lymph nodes cells were iso
15、lated and analyzed.The percentage of CD44high,SIINFEKL-specific CD8 T cells was assessed by three-color FACS staining with SIINFEKL MHC class I tetramer and antibodies to CD8 and CD44.Plots shown are gated on the SIINFEKL tetramer positive lymphocytes;values are mean percentages of CD44 cells within
16、 the SIINFEKL tetramer-positive population.,CD8+Memory T Cells Need Antigen-specific CD4+T-helper Cells to Achieve Tumor Protection,Ovalbumin CTL epitope(SIINFEKL)-specific T cells were parked in Rag-/-mice for 42 days to generate mCTL and then adoptively transferred to C57BL/6 mice.Two groups of re
17、cipient C57BL/6J mice were immunized 8 days prior with 50 ug of OVT in incomplete Freunds adjuvant or 20 ug keyhole limpet hemocyanin(KLH)protein as control in incomplete Freunds adjuvant.Mice were challenged with ovalbumin-expressing tumor cells(EG7)in the scruff of the neck 1 day after adoptive tr
18、ansfer.Normal C57BL/6 control mice were challenged with EG7 without any treatment.,Absence of tumor protection in mice without antigen-specific T-helper cells is not because of lower levels of tumor antigen(SIINFEKL)-specific CD8+T cells.,mCTLs were transferred to groups of C57BL/6 mice with or with
19、out immunization 8 days prior with 50 ug of OVT or with 20 ug of KLH protein control.C57BL/6 control group was given no treatment.IFN-Elispot assays were performed using the splenocytes of these mice,which were challenged in vitro with(A)1 ug/ml SIINFEKL peptide or(B)8ug/ml OVA T-helper peptide to d
20、etermine the presence of ovalbumin-specific CD8+CTLs and CD4+T-helper cells.,In contrast to mCTLs,eCTLs do not need T help to kill tumor.,eCTLs generated from RAG-/-mice were transferred at(A)day 7 or(B)day 14 to C57BL/6 mice that had been immunized 8 days earlier with 50 ug of OVT or as control 20
21、ug of KLH.C57BL/6 mice without any treatment were used as controls(EG7 and EL4 controls).Ovalbumin-expressing tumor cells(EG7)were injected under scruff of the neck of some groups of mice at(A)day 7 or(B)day 14.The parent tumor cell line EL4 that does not contain ovalbumin gene was injected into oth
22、er groups of mice at day7(A)as nonspecific tumor control.,Study of the long-lived memory CD8+T cells generated in C57BL/6J mice.,Ovalbumin CTL epitope(SIINFEKL)-specific OT-1 cells were adoptively transferred to syngeneic C57BL/6 mice;mice were immunized with SIINFEKL/CFA to activate ovalbumin-speci
23、fic CD8+T cells,and the mice were kept for 100 days to ensure that all transferred OT-1 are of long-lived memory phenotype before challenging with tumor.Eight days before tumor challenge,two groups of mice were immunized with 50 ug of OVT in incomplete Freunds adjuvant.Untreated C57BL/6 mice were al
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