免疫学技术进展 及应用 王晓健 浙江大学免疫学研究所 wangxiaojian@cad.zju.edu.cn 0571-88208280

酶联免疫吸附试验（ELISA）

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

Antigen-Antibody Interaction 一 抗原抗体的检测 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

Traditional Immunoassays 一 抗原抗体反应 Agglutination(aggregation) Assays: Immunodiffusion Complement Fixation EIA (IHC/ELISA/ELISPOT) Immunofluorescence (IFA FACS) ICS （Intracellular cytokine staining） CLIA （Chemiluminescence immumoassay） Traditional Immunoassays Modern Immunoassays 酶免疫 荧光免疫分析 （FIA） 化学发光免疫分析 （CLIA）

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。

标记抗体 horseradish peroxidase IHC Western Blot fluorescence FACS CD4 CD8 IHC Western Blot fluorescence FACS microscope   

免疫标记技术 EIA (Enzyme Immunoassay) Color Detection Substrate

IHC

第二抗体 One conjugated 2nd antibody can detect all primary antibodies with same isotype, which reduce the labor to conjugate all primary antibodies 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. Specific 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.

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。

酶联免疫吸附试验（ELISA） 夹心法（测抗原） 间接法（测抗体）

ELISA 当需要检测无法获得两种以上单一性抗体的抗原，或是不易得到足够的纯化抗体以固著于孔盘上时

ELISA 亲和素是一种糖蛋白，一分子亲和素可以与四个生物素小分子发生专一亲和作用，类似抗原与抗体亲和。生物素很易与蛋白质（如抗体等）以共价键结合。这样，结合了酶的亲和素分子与结合有特异性抗体的生物素分子产生反应，既起到了多级放大作用，又由于酶在遇到相应底物时的催化作用而呈色。

ELISA间接法测抗体

ELISPOT 酶联免疫斑点检测技术（Enzyme-linked immuno-sorbent spot，ELISPOT) ，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1/106，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

ELISA 和ELISPOT 比较 ELISA通过显色反应，在酶标仪上测定吸光度，与标准曲线比较得出定量的可溶性蛋白总量。 ELISPOT通过显色反应，在细胞分泌这种可溶性蛋白的相应位置上显现清晰可辨的斑点，可直接在显微镜下人工计数斑点或通过ELISPOT分析系统对斑点进行计数，1个斑点代表1个活性细胞，从而计算出分泌该蛋白或者细胞因子的细胞的频率。 单细胞水平检测，ELISPOT比ELISA和有限稀释法等更灵敏，能从 20万-30万 细胞中检出 1个 分泌该蛋白的细胞

免疫荧光技术 Immunofluorescence） CD4 CD8

Immunofluorescence An Example

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）

时间分辨荧光免疫测定(time-resolved fluoresence immunoassay,TR-FIA) 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，由于镧系元素在特定波长的紫外线激发下，从基态跃迁到高能态，有固定的时间延迟。 由于时间延迟以及激发光与发射光的频移，有效的屏蔽了由血清等生物物质产生的荧光，降低了本底。

抗体的应用：Western blotting 先将抗原经SDS-PAGE电泳分开，再将分开的蛋白质转印到醋酸纤维膜上，然后放入含有抗体的溶液中，洗去未结合抗体，再加标记的二抗，通过显色或发光显示结果。

抗体的应用 Immunoprecipitation. A protein mixture is incubated with specific antibody. Any antigen–antibody complexes that form are precipitated from solution by the addition of Protein A-coated 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 specific 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 specifi c antigen to the immobilized antibody. Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing. A solution with the appropriate pH and salt concentration to disrupt Ag–Ab binding is then passed through the column to elute (wash off) the antigen of interest.

二、细胞免疫技术 Lymphocyte Function Assays Isolation of lymphocytes Function Measurement --Proliferation --Phagocytosis --Cytokine --DTH --Apoptosis

外周血单个核细胞的分离 葡聚糖-泛影葡胺密度梯度离心法: Before centrifuge After centrifuge Fig.1

免疫磁珠分离法 （immune magnetic bead, IMB） 将已知抗细胞表面标记的抗体交联在磁性微珠上，与细胞悬液作用后置于磁场中，借磁力的作用将磁珠结合的细胞分开。

Identification of cell subsets by FACS Type of cell CD markers stem cells CD34+,CD31- all leukocyte groups CD45+ Granulocyte CD45+,CD15+ Monocytes CD45+,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- B cell T cell CD4+ T cell CD8+ Tcells Tregs (CD4+CD25+) Conventional CD4 Immune Cell types and subtypes defined by surface markers (CDs)

Basics of Flow Cytometry Fluidics Cells in suspension flow in single Optics scatter light and emit fluorescence that is collected, filtered Electronics converted to digital values Stored & analyzed on a computer 流式细胞仪的工作原理是将待测细胞经特异性荧光染料染色后放入样品管中，在气体的压力下进入充满鞘液的流动室。在鞘液的约束下细胞排成单列由流动室的喷嘴喷出，形成细胞柱，后者与入射的激光束垂直相交，液柱中的细胞被激光激发产生荧光。仪器中一系列光学系统(透镜、光阑、滤片和检测器等)收集荧光、光散射、光吸收或细胞电阻抗等信号，计算机系统进行收集、储存、显示并分析被测定的各种信号，对各种指标做出统计分析 *Collection *Drop charged and collected

FACS Flow cytometry or Fluorescence Activated Cell Sorter (FACS) Quantitative, multi- parameter analysis of large numbers of individual cells Cell surface markers Intracellular proteins Ca++ mobilization 12 colors and 15 parameters Sorting: 70,000 cell/second

FCM 前向角散射光信号FSC 侧向角散射光信号SSC

CTL cytotoxic T lymphocyte MLR DTH （Delayed Type Hypersersitivity） T cell proliferation CTL cytotoxic T lymphocyte MLR DTH （Delayed Type Hypersersitivity）

T cell proliferation Morphology 3H-thymidine labeling MTT FACS-CFSE staining CFSE能够轻易穿透细胞膜，在活细胞内与胞内蛋白共价结合，水解后释放出绿色荧光。在细胞分裂增殖过程中，它的荧光强度会随着细胞的分裂而逐级递减

T cell proliferation Morphology

T cell proliferation 3H-thymidine labeling

T cell proliferation -MTT Mitochondria enzyme catalyze the reduction of MTT – Turn blue

T cell proliferation FACS-CFSE staining

CTL Assay

CTL Assay Supernatant

51Cr Release Assay Controls: Natural release well – medium; Maximal release well – Detergent Lysed No antigen control Control release ratio (》5%): average value Control cpm– Natural release Maximal release – Natural release Specific release ratio(》10%): Specific cpm– Natural release Natural release ratio(《15%)

CTL – DIOC/PI staining

Tetramer MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells). Biotinylated recombinant class I molecules folded with the peptide of interest and β2M 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. 由于可溶性MHC单体分子与TCR的亲和力很低，解离快，而多价分子可与一个特异性T细胞上的多个TCR结合，使其解离速度大大减慢

Tetramer Tetramers can bind to three TCRs at once, allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction.

Utility of Tetramer CD8 response related(now CD4 too) and known epitope Infectious Disease HIV-AIDS, EBV, CMV, HPV, HBV, HCV, Influenza, Measles, Malaria, TB and RSV Tumor Immunology Breast, Prostate, Melanoma, Colon, Lung, Cervical, Ovarian and Leukemia Autoimmune Disease Diabetes, Lyme disease, Multiple sclerosis, Rheumatoid arthritis, Autoimmune vitiligo Transplantation EBV and CMV

NP 118-126 LCMV 的抗原部位

DTH In vivo Immunization Challenge (Ear/Foogpad) IV 超敏反应，抗原特异性T细胞介导 ，需要效应分子的合成，进程比较缓慢。是研究细胞免疫功能的常用动物模型。主要是研究T 细胞的功能。 Measurement (Calipers)

PFC Plaque forming cell Antibody production In VIVO B cell proliferation PFC Plaque forming cell Antibody production In VIVO

B cell proliferation

PFC Plaque forming cell

In Vivo

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

Apoptosis detection In the TUNEL assay, fragmented DNA is labeled by terminal deoxynucleotidyl transferase (TdT) to reveal apoptotic cells. 细胞在发生凋亡时，会激活一些DNA内切酶，这些内切酶会切断核小体间的基因组DNA。细胞凋亡时抽提DNA进行电泳检测，可以发现180-200bp的DNA ladder。基因组DNA断裂时，暴露的3’-OH可以在末端脱氧核苷酸转移酶(Terminal Deoxynucleotidyl Transferase, TdT) 的催化下加上荧光素 (FITC) 标记的dUTP (fluorescein-dUTP)

细胞因子检测 Real-time PCR (mRNA Level) ELISA/ELISPOT Intracellular staining (FACS) 4.Biological Function

Intracellular Staining Identification of different T helper cells characterized by different cytokine production

HLA检测 血清学方法：微量细胞毒试验或补体依赖的细胞毒试验（CDC） 细胞学方法：混合淋巴细胞反应 分子生物学方法:PCR

HLA检测 单链构象多态性检测是一种基于DNA构象差别来检测点突变的方法。相同长度的单链DNA，如果碱基序列不同，形成的构象就不同，这样就形成了单链构象多态性。

HLA检测

Another man: A2, Aw24; B7, Bw54; Cw3 HLA检测 亲子鉴定实例： Wife: A2, A11; B27, Bw44 Child 1: A2, A1; Bw44, B15, Cw3 Child 2: A2, Aw24; Bw44, Bw54; Cw3 Husband: A1, A3; B15, -; Cw3 Another man: A2, Aw24; B7, Bw54; Cw3

人体免疫功能评价

人体免疫功能评价

应用举例 某课题组用幽门螺旋杆菌的尿素酶疫苗经黏膜途径免疫小鼠，请设计实验检测免疫后小鼠的免疫学功能？

应用举例 体液免疫功能： 特异性IgA抗体，特异性IgG抗体 细胞免疫功能 体外：T细胞增殖能力 产生细胞因子谱 CTL活性 免疫保护作用

Thank you ！