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1、免疫学技术 类型 抗原抗体反应 细胞免疫 水平 细胞 分子 基因 免疫学技术 Antigen-Antibody Interaction 抗原与抗体发生结合反 应的物质基础是抗原的 抗原决定基与抗体的抗 原结合部位之间的结构 互补性，二者相互结合 一 抗原抗体的检测 Agglutination(aggregation) Assays: Immunodiffusion Complement Fixation EIA (IHC/ELISA/ELISPOT) Immunofluorescence (IFA FACS) ICS （Intracellular cytokine staining） CLIA
2、 （Chemiluminescence immumoassay） Traditional Immunoassays 一 抗原抗体反应 Modern Immunoassays 凝集反应 扩散试验 补体结合反应 1，免疫标记技术 免疫标记技术是用荧光素、酶或放射性核 素等标记物标记抗体或抗原，进行的抗原 抗体反应，是目前应用最广泛的免疫学检 测技术。 标记物与抗体或抗原连接后不改变后者的 免疫特性，不仅提高了方法的灵敏度，而 且还具有快速、可定性或定量，甚至定位 等优点。 IHC Western Blot fluorescence FACS microscope CD4 CD8 标记抗体 hors
3、eradish peroxidase 免疫标记技术 EIA (Enzyme Immunoassay) Substrate Color Detection IHC 第二抗体 A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications. Spe
4、cific secondary antibodies are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred. One conjugated 2nd antibody can detect all primary antibodies with same isotype, which reduce the labor to conjugat
5、e all primary antibodies 第二抗体 酶免疫测定 酶免疫测定是用酶标记的抗体进行的抗原 抗体反应。 常用于标记的酶有辣根过氧化物酶 (horseradish peroxidase, HRP)、碱性磷酸 酶(alkaline phosphatase, AP)等。 常用的方法有酶联免疫吸附试验和酶免疫 组化法，前者测定可溶性抗原或抗体，后 者测定组织或细胞中的抗原。 ELISA ELISA C. Sandwich ELISA测抗原 D. ELISA间接法 测抗体 ELISPOT 酶联免疫斑点检测技术（Enzyme-linked immuno-sorbent spot，ELIS
6、POT) ，由Cecil Czerkinsky 在1983建立。 原理：用抗体捕获培养中细胞所分泌的细 胞因子，并以酶联斑点显色方式将其表现 出来。 ELISPOT 优点 其一，灵敏度高。检测水平1/1000000，是 目前为止，最灵敏的检测技术，灵敏度比 传统的ELISA 方法高2-3 个数量级。 其二，单细胞水平，活细胞功能检测。 其三，操作简便经济，可以进行高通量筛 选。 Two cytokines can be detected simultaneously CD4 CD8 免疫荧光技术 Immunofluorescence） Immunofluorescence An Example
7、 荧光染料 异硫氰酸荧光素（FITC）,藻红蛋白（PE）多甲藻叶绿素（PerCP）,异藻蓝蛋白 （APC） 荧光免疫技术 时间分辨荧光免疫测定(time-resolved fluoresence immunoassay,TR-FIA) 利用具有双功能基团结构的螯合剂，将镧系元素标 记到抗体（或抗原）上，经免疫反应形成复合物，由于镧 系元素能发出荧光，故分离除去未结合的成分后，利用时 间分辨荧光仪即可测定荧光强度，从而推测待测物含量。 荧光分析与普通分光不同，光电接受器与激发光不在 同一直线上，激发光不能直接到达光电接受器，从而大幅 度地提高了光学分析的灵敏度。但是，当进行超微量分析 的时候，激
8、发光的杂散光的影响就显得严重了。 但普通的荧光标志物荧光寿命非常短，激发光消失， 荧光也消失。不过有非常少的稀土金属（Eu、Tb、Sm、 Dy）的荧光寿命较长，可达12ms. 抗体的应用：Western blotting 抗体的应用 Immunoprecipitation. A protein mixture is incubated with specific antibody. Any antigenantibody complexes that form are precipitated from solution by the addition of Protein A-coated
9、beads that bind to the antibodies and collect at the bottom of the tube under the force of centrifugation. After washing, the desired antigen is released from the antibody-bound beads using altered pH and/or high salt concentration 抗体的应用 Affinity Chromatography Agarose beads bearing immobilized spec
10、ifi c antibody are placed into a column with a semi-permeable plug at the bottom. A solution containing antigen is passed slowly through the column, allowing the binding of specific antigen to the immobilized antibody. Unbound entities pass through the plug and any molecule that binds to the beads n
11、on-specifi cally is removed by extensive washing. A solution with the appropriate pH and salt concentration to disrupt AgAb binding is then passed through the column to elute (wash off) the antigen of interest. Lymphocyte Function Assays Isolation of lymphocytes Function Measurement -Proliferation -
12、DTH -Apoptosis -Phagocytosis -Cytokine 二、细胞免疫技术 Isolation of lymphocytes 盘选盘选(Panning)(Panning)分离法分离分离法分离T T细胞亚群细胞亚群 聚苯乙烯表面可以用作亲和性试剂的不溶性基质,以分离 淋巴细胞亚群.聚苯乙烯培养皿首先用纯化的羊抗鼠IgG抗 体包被,然后将鼠抗人(如抗CD4或抗CD8单抗)与T细胞反 应的细胞悬液加入培养皿中,孵育一段时间后,对CD4或 CD8抗原特异的T细胞即结合到平皿表面,而CD8+或CD4+ 细胞不结合,轻吸出未贴壁的细胞悬液,洗下粘附细胞。 Cell subsets Pu
13、rification Identification of cell subsets by FACS Immune Cell types and subtypes defined by surface markers (CDs) B cell T cell CD4+ T cell CD8+ Tcells Tregs (CD4+CD25+) Conventional CD4 Type of cell CD markers stem cells CD34+,CD31- all leukocyte groups CD45+ Granulocyte CD45+,CD15+ Monocytes CD45+
14、,CD14+ T lymphocyte CD45+,CD3+ T helper cell CD45+,CD3+,CD4+ Cytotoxic T cell CD45+,CD3+,CD8+ B lymphocyte CD45+,CD19+ or CD45+,CD20+ Thrombocytes CD45+,CD61+ Natural killer cell CD16+,CD56+,CD3- Fluidics Cells in suspension flow in single Optics scatter light and emit fluorescence that is collected
15、, filtered Electronics converted to digital values Stored Maximal release well Detergent Lysed No antigen control 51Cr Release Assay CTL DIOC/PI staining MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells). Biotinylated recombinant class I molecules
16、folded with the peptide of interest and 2M and tetramerized by a fluorescently labeled streptavidin(Streptavidin binds to four biotins per molecule.) . This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex. Antigen specific responses can be measured as CD8+, tetramer+ T cells as a fraction of all CD8+ lymphocytes.
