第七章抗原抗体反应及应用教材课件.ppt

第七章 抗原抗体反应及应用,第一节 抗体的制备,多抗（ployclonal antibody）单抗（monoclonal atibody）抗体库（antibody library）基因工程抗体（engineering antibody）催化抗体（catalytic antibody or abozyme）抗血清：含有特异性抗体的动物血清。,一、抗血清的制备,1、免疫动物 抗原 佐剂 免疫动物2、抗血清的纯化和保存 采血 抗血清的纯化保存3、抗血清的特性鉴定 滴度（titer） 亲和（合）力 特异性,二、抗体的特性,1 抗原-抗体反应作用力,抗原抗体作用力,非共价键结合,氢键 离子键 范德华力 疏水键,2 抗体的特性测定,滴度（Titer）是指在给定条件下，结合一定量抗原所需的最大的抗血清稀释倍数。（因方法不同有差异）特异性（Specificity） 特异性检测/竞争性检测亲和力（Affinity）和亲合力（Avidity）,亲和力是单个抗原表位与单个抗体结合位点之间的作用力，反映抗体与抗原表位特异性结合强弱的量化指标。,Calculation of Affinity,Ag + Ab Ag-Ab,Applying the Law of Mass Action:,Reversible,亲合力（Avidity）p156,多价抗原与抗体结合强度的总体评价 ，反映抗原抗体结合的总体效果。（提高浓度改善抗原抗体亲和性）,3 影响抗原-抗体反应的因素,亲和力亲合力抗原抗体比例,抗原的物质形式环境条件 温度、离子浓度等,Polyclonal antibodies can form lattices, or large aggregates, that precipitate out of solution.If each antigen molecule contains only a single epitope recognized by a given monoclonal antibody, the antibody can link only two molecules of antigen and no precipitate is formed.,第二节 抗原抗体反应原理,抗原抗体反应是非共价的特异性可逆反应。 体外免疫检测的原理。一、抗原抗体反应的热力学与动力学 1 溶液中抗原抗体反应的化学平衡 Ag+Ab=Ag-Ab K = SL /SL= K+/K- p152 K : 平衡常数或内在亲和力。Kd:解离常数,达到反应平衡需要的时间较长，但反应进行90%很容易。K相同并不能说明K+和K-的大小。 K +大时，有利于结合，抗原抗体结合稳定 K-大时， 达到平衡点快，结合不稳定 。 前者用于标记，后者用于亲和层析。,抗体结合抗原的特异性,同源抗原（强）异源抗原（弱）变态抗体（强）抗原末端或表面基团具有免疫优势三、抗体与大分子多价抗原的反应1 多抗原决定簇-大分子抗原特征 抗原决定簇（表位）-多价抗原-表位重叠,半抗原为单抗原决定簇结合在固体上具有多抗原决定簇的效果与液体中的半抗原反应不同2 抗原抗体的亲和力和亲合力亲和力(affinity)单价抗原与抗体结合的强弱亲合力(avidity) 多价抗原与抗体结合强度的总体评价 3 抗体与多价抗原结合的浓度带现象 抗体与单价抗原结合往往形成可溶性复合物,抗体与多价抗原可以形成沉淀（条件） 抗原抗体反应时 前带区（抗体过量，沉淀少） 等带区（抗体抗原适量，大量沉淀） 后带区（抗原过量，沉淀少） 初级反应（未出现沉淀） 次级反应（出现沉淀）,形成沉淀需要一定时间，另外温度、pH值、离子强度、补体等沉淀的形成可以用网络理论解释4 交叉反应 抗体与同源抗原反应，又与异源抗原结合就产生了交叉反应。同源抗原结合强，异源结合弱。,交叉反应,单个抗体结合位点能与不同的抗原表位发生反应或者一个抗体能与不同的抗原反应。 当抗原具有相同或相似的抗原表位时即可发生交叉反应。,第三节 常见免疫分析方法,利用抗原抗体特异性反应特点，对其中之一进行定性定量分析的方法。特异、灵敏、迅速特点。一、免疫沉淀（immune precipitation),沉淀反应： 可溶性抗原（血清蛋白、细胞裂解液等）与相应抗体结合后，在有电解质存在时，比例适当，出现肉眼可见的沉淀物。 半固体琼脂凝胶为介质。,A precipitation curve for a system of one antigen and its antibodies,A zone of antibody excess- precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant; an equivalence zone of maximal precipitation - antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant; a zone of antigen excess - precipitation is inhibited and antigen not bound to antibody can be detected in the supernatant.,Precipitation reactions（沉淀反应）（soluble) in fluids yield a precipitin curve,前带区（抗体过量，沉淀少） 等带区（抗体抗原适量，大量沉淀） 后带区（抗原过量，沉淀少）形成沉淀需要一定时间，另外温度、pH值、离子强度、补体等,1 溶液中的沉淀反应 环状沉淀试验,抗血清,沉淀线,抗原,在凝胶或琼脂中形成可见的沉淀线,2. 两种类型免疫扩散,单向扩散（radial immunodiffusion）双向扩散（double immunodiffusion） 。,单向扩散(radial immunodiffusion),双向扩散(double immunodiffusion),Single diffusion,原理:检测抗原,单向免疫扩散：,打孔,观察,（含量）,（2）双向免疫扩散：,琼脂,Ab,免疫电泳,血清免疫电泳鉴定-特异图谱火箭免疫电泳对流免疫电泳,（1）免疫电泳原理：电泳后双扩,应用：血清蛋白种类分析,An antigen preparation (orange) is first electrophoresed, which separates the component antigens on the basis of charge. Antiserum (blue) is then added to troughs on one or both sides of the separated antigens and allowed to diffuse; In time, lines of precipitation (colored arcs) form where specific antibody and antigen interact.,Immunoelectrophoresis Combines Electrophoresis and Double Immunodiffusion,Rocket diffusion,antibody,(3)对流免疫电泳：,方法和原理：双扩电泳,Immunoprecipitation using magnetic bead to precipitate antigen,4. 免疫共沉淀,5. 免疫凝集反应(Agglutination Reactions),抗体与颗粒抗原结合形成的可见聚集反应。The interaction between antibody and a particulate antigen results in visible clumping called agglutination,Hemagglutination (血细胞凝集）Is Used in Blood Typing,ABO血型系统特点：,Passive Agglutination Is Useful with Soluble Antigens,Agglutination reactions employing synthetic beads can be read rapidly, often within 3 to 5 minutes of mixing the beads with the test sample.,can detect small amounts of antibody (concentrations as low as nanograms per milliliter).,Definition - agglutination test done with a soluble antigen coated onto a particle,A modification of the agglutination reaction, called agglutination inhibition, provides a highly sensitive assay for small quantities of an antigen.凝集抑制,In Agglutination Inhibition, Absence ofAgglutination Is Diagnostic of Antigen（eg. Bacteria ),The original home pregnancy test kit employed hapten inhibition to determine the presence or absence of human chorionic gonadotropin (HCG人绒毛膜促性腺激素).,Pregnancy,Mechanism of home pregnant test kit,间接凝集抑制试验:乳胶妊娠试验,将待测样品中的抗原与已知抗体作用后，再与相应抗原乳胶颗粒混合。因没有游离抗体的存在，乳胶颗粒表面的抗原不能与抗体结合出现凝集现象，即凝集被抑制。孕妇尿液中含HCG，用抗HCG与之结合后，再加HCG乳胶，则不出现凝集现象。,方法 1、取玻片一张，左侧加生理盐水一滴，右侧加待检尿液一滴。 2、两侧各加抗HCG（HCG诊断血清）一滴，混匀2分钟。 3、两侧各加HCG乳胶抗原一滴，混匀2分钟，观察结果。,Agglutination inhibition assays can also be used to determine whether an individual is using certain types of illegal drugs, such as cocaine or heroin.,Agglutination inhibition assays are widely used in clinical laboratories to determine whether an individual has been exposed to certain types of viruses that cause agglutination of red blood cells.,二、免疫标记、放射免疫分析,放射免疫测定法（RIA）： 131I、125I标记，微量物质测定,或放射自显影,酶联免疫吸附试验EIA（Enzyme immunoassay）ELISA （Enzyme-linked immunosorbent assay） 直接法 包被抗体-标记抗原 包被抗原-标记抗体,ELISA原理： 将已知抗原或抗体吸附于固相载体（聚苯乙烯微量反应板）表面，使抗原抗体反应在固相表面进行，用洗涤液将游离成分洗去。,间接法 包被抗原 - 一抗 - 标记二抗夹心法直接夹心 ：包被抗体 - 抗原 - 标记抗体 间接夹心：包被抗体 - 抗原 - 一抗 - 标记二抗Dot-ElISA,*间接法： 检测抗体,方法：*双抗体夹心法： 检测抗原,包被,标本,洗涤,ELISPOT酶联免疫斑点测定法,3 荧光免疫分析,膜抗原,4 亲和标记免疫分析 生物素-亲和素（链亲和素）亲和力高 地高辛-抗体,Fig. 3. Northern blotting to detect DjPreb RNA. Lane 1, total RNA of planarians. 28 S and 18 S rRNA bands are indicated. Lane 2, the blot was hybridized with DIG-labeled DjPreb RNA probe.,Fig.4. Expression patterns of DjPreb mRNA in D.japonica juveniles detected by whole-mount in situ hybridization.,三、免疫定位分析 荧光免疫定位 酶标记免疫定位 免疫组化/免疫印迹,蛋白印迹Western Blotting,(a) separated by electrophoresis in PAGE/SDS-PAGE, which separates the components according to their molecular weight; lower molecular weight components migrate farther than higher molecular weight ones. (b) The gel is removed from the apparatus and applied to a protein-binding sheet of nitrocellulose or nylon and the proteins in the gel are transferred to the sheet by the passage of an electric current. (c) Addition of enzyme-linked antibodies detects the antigen of interest. (d) The position of the antibodies is visualized by means of an ELISA reaction that generates a highly colored insoluble product that is deposited at the site of the reaction.,免疫印迹Western Blot,His-Pv重组蛋白表达SDS-PAGE分析结果。M，marker；1，经过诱导的带有重组质粒His-Pv超声后沉淀；2，经过诱导的带有重组质粒His-Pv超声后上清；3，未经诱导的带重组质粒His-Pv的菌。,斑马鱼卵无细胞体系中C3和Bf的Western blot检测（M：marker；H：人血清；Z：斑马鱼卵无细胞体系）兔抗人C3多克隆抗体和羊抗人Bf多克隆抗体与人血清和斑马鱼卵无细胞体系均有反应。,Western blot 结果表明兔抗人C3多克隆抗体和羊抗人Bf多克隆抗体与人血清和斑马鱼卵无细胞体系均有反应（图2-20）。兔抗人C3抗体与斑马鱼卵无细胞体系形成了三条特异性条带，其中一条带对应的分子量约为185 kD，与人C3的分子量大小相当；此外，还有两条分子量约为115 kD和70 kD的条带，分别与C3和C3的大小相一致。斑马鱼卵无细胞体系与羊抗人Bf抗体的反应仅产生了一条特异性条带，分子量与人的Bf很接近，约为93 kD。这一结果说明在斑马鱼卵子中确实存在参与替代途径的补体成分。,Fig. 7 Localization of AmphiTIP30 transcripts in different tissues of adult amphioxus. (A) A micrograph shows the presence of AmphiTIP30 transcripts in the female amphioxus and predominantly expressed in the ovary. (B and C) A micrograph shows the absence of AmphiTIP30 in control sections. (D) Micrographs show the absence of AmphiTIP30 in the spermary in amphioxus. Scale bars represent 100 lm.,Fig. 5. Ectopic dorsal expression of ventral markers and CNS duplication after Smed-BMP and Smed-Smad1 RNAi. (A, D) Smed-eye53 expression in controls. In panel D white arrows indicate the pharynx neural plexus and black arrows indicate the visual cells. After RNAi, the treated animals show ectopic dorsal Smed-eye53 expression (arrowheads in panels E and F). Black arrows in panels EF indicate the eyes. Red arrow in panel E points to an extra eye. (GR) Immunostaining of the planarian nervous system with anti-SYNORF1 in control (G, J, M,), Smed-BMP (H, K) and Smed-Smad1 (I, L) RNAi-treated planarians. Control animals have a normal ventral CNS (G) and dorsal submuscular plexus (J). Partial nerve cords are formed on the dorsal side of treated animals at the level of the submuscular plexus (arrows in panels K and L).,Immunostaining,Animals were killed in 2% HCl for 5 min on ice and then fixed in Carnoys solution for 2 h at 4 C. anti-SYNORF1, a mouse monoclonal antibody specific for synapsin (Developmental Studies Hybridoma Bank, used at a dilution of 1:25). Goat anti-mouse secondary antibody conjugated to Alexa 488 (Molecular Probes) was used at a 1:400 dilution. Samples were mounted in SlowFade Gold antifade reagent (Invitrogen). Confocal laser scanning microscopy was performed with a Leica TCS 4D (Leica Lasertechnik, Heidelberg) adapted for an inverted microscope (Leitz DMIRB).,四、其他应用,免疫亲和层析生物传感器,生物受体,转导系统,信号放大并检测,练习题,概念抗血清 2. 滴度 3. 亲和力 4.亲合力简答如何制备抗体（过程）？列举抗原抗体的应用实例及原理？沉淀（凝集）反应； ELISA、蛋白印迹等,