免疫学检测与免疫学技术.ppt

Chapter 10. 免疫学检测方法与免疫技术 一、概述 免疫学检验方法和免疫化学技术主要包括： 抗原抗体的制备、纯化和鉴定，免疫扩散、免疫电泳、免疫凝集试验、补体结合试验，免疫细胞分离、纯化和鉴定，免疫功能检测，细胞因子检测，放射免疫检测，免疫酶标检测，荧光和发光免疫技术，免疫组化实验方法，原位杂交免疫组化，免疫PCR技术，免疫微球的应用，免疫电镜技术，细胞凋亡的检测方法，膜受体分析，胞内钙镁浓度的测定和细胞间通讯，流氏细胞仪技术及应用等。 从上述内容可以看出，免疫技术是免疫学和物理、化学及电子信息和分子生物学理论和技术的结合产物。其应用涉及生命科学的各个领域，已成为现代医学和生物学研究工作不可缺少的有效工具。,免疫技术的原理和特点： 基于免疫应答的理论，即抗原抗体的特异性反应。基于免疫细胞的结构、应答特性和分子基础。 具有特异性 高度灵敏性； 可重复性；广泛适用性；快速反应性；可观察性；可定性、定量，即可控性；组化定位特性等特点。 概括起来可分为： 沉淀反应，即可溶性抗原和抗体间的反应。 凝集反应，即颗粒性抗原和抗体间的反应。 免疫标记，即用酶、同位素、荧光素或电子致密物质标记。 免疫印渍，即用标记抗体与待测蛋白质印迹结合。 单抗及工程抗体技术，即分子生物技术。 流氏细胞术，即荧光标记，流体喷射，激光和能谱检测，电 脑分析。,Ag-Ab reactions Tests for Ag-Ab reactions,Nature of Ag/Ab Reactions,Lock and Key Concept,Non-covalent Bonds,Hydrogen bonds Electrostatic bonds Van der Waal forces Hydrophobic bonds,Reversible,Multiple Bonds,Affinity = attractive and repulsive forces,Affinity,Strength of the reaction between a single antigenic determinant and a single Ab combining site,Calculation of Affinity,Ag + Ab Ag-Ab,Applying the Law of Mass Action:,Avidity,The overall strength of binding between an Ag with many determinants and multivalent Abs,Specificity,The ability of an individual antibody combining site to react with only one antigenic determinant. The ability of a population of antibody molecules to react with only one antigen.,Cross Reactivity,The ability of an individual Ab combining site to react with more than one antigenic determinant. The ability of a population of Ab molecules to react with more than one Ag,Factors Affecting Measurement of Ag/Ab Reactions,Affinity,Avidity,Ag:Ab ratio,Physical form of Ag,Tests Based on Ag/Ab Reactions,All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes All tests based on Ag/Ab reactions can be used to detect either Ag or Ab,Agglutination Tests,Lattice Formation,Agglutination/Hemagglutination,Definition - tests that have as their endpoint the agglutination of a particulate antigen Agglutinin/hemagglutinin,Agglutination/Hemagglutination,Quantitative agglutination test Titer Prozone,Agglutination/Hemagglutination,Definition Qualitative test Quantitative test,Applications Blood typing Bacterial infections Fourfold rise in titer,Practical considerations Easy Semi-quantitative,Passive Agglutination/Hemagglutination,Definition - agglutination test done with a soluble antigen coated onto a particle,Applications Measurement of antibodies to soluble antigens,Coombs (Antiglobulin)Tests,Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes,Coombs (Antiglobulin)Tests,Indirect Coombs Test Detects anti-erythrocyte antibodies in serum,Coombs (Antiglobulin)Tests,Applications Detection of anti-Rh Ab Autoimmune hemolytic anemia,Agglutination/Hemagglutination Inhibition,Definition - test based on the inhibition of agglutination due to competition with a soluble Ag,Agglutination/Hemagglutination Inhibition,Applications Measurement of soluble Ag Practical considerations Same as agglutination test,Definition,Precipitation Tests,Lattice Formation,Radial Immunodiffusion (Mancini),Interpretation Diameter of ring is proportional to the concentration Quantitative Ig levels,Method Ab in gel Ag in a well,Immunoelectrophoresis,Method Ags are separated by electrophoresis,Interpretation Precipitin arc represent individual antigens,Ab is placed in trough cut in the agar,Immunoelectrophoresis,Method Interpretation Qualitative Relative concentration,Countercurrent electrophoresis,Method Ag and Ab migrate toward each other by electrophoresis Used only when Ag and Ab have opposite charges,Qualitative Rapid,Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (ELISA),Lattice formation not required,Competitive RIA/ELISA for Ag,Method Determine amount of Ab needed to bind to a known amount of labeled Ag,Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor,Competitive RIA/ELISA for Ag,Method cont. Determine amount of labeled Ag bound to Ab NH4SO4 anti-Ig Immobilize the Ab,Quantitative Most sensitive test,Concentration determined from a standard curve using known amounts of unlabeled Ag,Solid Phase Non-Competitive RIA/ELISA,Ab detection Immobilize Ag Incubate with sample Add labeled anti-Ig Amount of labeled Ab bound is proportional to amount of Ab in the sample,Quantitative,Solid Phase Non-Competitive RIA/ELISA,Ag detection Immobilize Ab Incubate with sample Add labeled antibody Amount of labeled Ab bound is proportional to the amount of Ag in the sample,Quantitative,Tests for Cell Associated Antigens,Lattice formation not required,Immunofluorescence,Direct Ab to tissue Ag is labeled with fluorochrome,Immunofluorescence,Indirect Ab to tissue Ag is unlabeled Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab.,Qualitative to Semi-Quantitative,Immunofluorescence,Flow Cytometry Cells in suspension are labeld with fluorescent tag Direct or Indirect Fluorescence Cells analyzed on a flow cytometer,Immunofluorescence,Flow Cytometry cont. Data displayed,Green Fluorescence Intensity,Number of Cells,Unstained cells,FITC-labeled cells,One Parameter