第四章 抗原抗体反应及应用 抗血清：指抗原人工免疫实验动物，获得含 特异性抗体的血清。 多克隆抗体 (PAb)： 多个抗原决定簇免疫机体

Presentation on theme: "第四章 抗原抗体反应及应用 抗血清：指抗原人工免疫实验动物，获得含 特异性抗体的血清。 多克隆抗体 (PAb)： 多个抗原决定簇免疫机体"— Presentation transcript:

1 第四章 抗原抗体反应及应用 抗血清：指抗原人工免疫实验动物，获得含 特异性抗体的血清。 多克隆抗体 (PAb)： 多个抗原决定簇免疫机体
第四章 抗原抗体反应及应用 第一节 抗体的制备 抗血清：指抗原人工免疫实验动物，获得含 特异性抗体的血清。 多克隆抗体 (PAb)： 多个抗原决定簇免疫机体 所产生的多种抗体的混合物。 单克隆抗体（monoclonal antibody，MAb） ： 只针对某一特定的抗原决定簇，纯度高的抗体。

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4 一、抗血清的制备 1、免疫动物 抗原 佐剂 免疫动物 2、抗血清的纯化与保存 采血 纯化与保存 3、抗血清的鉴定 效价 亲和力

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6 Affinity =  attractive and repulsive forces
Strength of the reaction between a single antigenic determinant and a single Ab combining site Ab Ag High Affinity Ab Ag Low Affinity Affinity =  attractive and repulsive forces

7 Calculation of Affinity
Ag + Ab  Ag-Ab Applying the Law of Mass Action: Keq = [Ag-Ab] [Ag] x [Ab]

8 二、单抗的制备 1、单抗的制备原理 2、单抗的制备方法 抗原提纯和动物免疫 骨髓瘤细胞及饲养细胞的制备 细胞融合 筛选阳性株
单克隆抗体的制备和冻存 单克隆抗体的纯化 GO

9 骨髓瘤细胞 脾脏淋巴细胞 （ HGPRT-TK- ) （HGPRT+TK+） 在HAT中不能生长 体外培养过程中死亡 ↓←PEG 杂交瘤细胞
骨髓瘤细胞 脾脏淋巴细胞 （ HGPRT-TK- ) （HGPRT+TK+） 在HAT中不能生长 体外培养过程中死亡 ↓←PEG 杂交瘤细胞 (HGPRT+、TK+) ↓ 在HAT中大量长期繁殖 克隆化 产生单抗 BACK

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12 Production of a Monoclonal Antibody
Most substances used for immunization consist of a mixture of antigens, resulting in a polyclonal antibody response directed against many different antigenic determinants.(2) A monoclonal antibody, however, is specific not just for the immunizing antigen, but also for a particular determinant on the antigen. Köhler and Milstein first described the production of monoclonal antibodies through hybridoma technology in 1975, for which they were subsequently awarded the Nobel Prize.(3) A mouse is repeatedly immunized with an antigen of choice, causing B cells to produce antibodies specific for that antigen. B cells from the immunized donor are fused to an immortalized myeloma cell whose own antibody synthesis has shut off. From this fusion, hybridomas producing antibodies with the desired characteristics (for example, antigen and epitope specificity, affinity, immunoglobulin class) can be selected to provide a homogeneous source of monoclonal antibody to the immunizing antigen. BACK

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18 三、单抗的应用 临床检测、抑制器官移植排斥、治疗自 身免疫疾病、生物导弹等。

19 Additional Cell-Killing Mechanisms of Monoclonal Antibodies
In addition to their potentially synergistic interactions with the host's immune effector cells, monoclonal antibodies may mediate direct tumor cell lysis.(4,7) Direct effects such as apoptosis (programmed cell death) and inhibition of cell proliferation have been demonstrated in vitro.(8) Other cell-killing mechanisms include the use of radionuclides or toxins conjugated to antibodies that target antigens on tumor cells. Radionuclides: Conjugation of a radionuclide to the antibody causes tumor destruction; however, harmful bystander effects occur to surrounding, healthy cells. Toxins: Toxins conjugated to antibodies produce a powerful antitumor effect due to the potency of the toxin. The nonspecific effects of these toxins on healthy tissues, however, can result in considerable toxicity.(4)

20 Antibody-Host Interactions in Cancer Immunotherapy
In order to maximize antibody binding to target molecules, an ideal antibody for cancer therapy should have a high affinity for, and a low dissociation rate from, its antigen.(4) Experience to date suggests that only a small fraction of injected antibody actually reaches the tumor.(5) An effective unconjugated antibody, therefore, should work synergistically with the host's immune effector mechanisms. In addition, it should be delivered in a nontoxic form. Therapeutic antibodies that induce effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytolysis(6) have the potential to provide targeted cancer therapy that is safe and effective without the use of potentially harmful conjugates such as toxins or radionuclides. The presence of a human Fc portion, as it appears on chimeric or humanized antibodies, is essential for efficient recruitment of human effector mechanisms.

21 第二节 抗原抗体反应的原理 一、抗原抗体反应的原理 1.亲水胶体转化疏水胶体 2.抗原抗体结合力 电荷引力（静电引力） 范登华引力
第二节 抗原抗体反应的原理 一、抗原抗体反应的原理 1.亲水胶体转化疏水胶体 2.抗原抗体结合力 电荷引力（静电引力） 范登华引力 氢键结合力 疏水作用力

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23 二、抗原抗体反应的特性 Nature of Ag/Ab Reactions
Specificity Lock and Key Concept antibody combining site to react with only one antigenic determinant. The ability of a population of antibody molecules to react with only one antigen.

24 Cross Reactivity Cross reactions
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 Anti-A Ab Ag C Similar epitope Cross reactions Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope

25 Non-covalent Bonds Multiple Bonds Reversible Hydrogen bonds
Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 Hydrogen bonds Electrostatic bonds Van der Waal forces Hydrophobic bonds Multiple Bonds Reversible

26 Factors Affecting Measurement of Ag/Ab Reactions
三、影响抗原抗体反应的因素 Factors Affecting Measurement of Ag/Ab Reactions Ab excess Ag excess Affinity Ag:Ab ratio Physical form of Ag Equivalence – Lattice formation PH Temperament Electrolyte

27 第三节 常见的免疫分析方法 一、凝聚反应(Agglutination) 二、沉淀反应 1、溶液中的沉淀反应 2、凝胶扩散沉淀 3、免疫电泳
第三节 常见的免疫分析方法 一、凝聚反应(Agglutination) 二、沉淀反应 1、溶液中的沉淀反应 2、凝胶扩散沉淀 3、免疫电泳 血清免疫电泳 火箭电泳 对流免疫电泳 三、补体参与的反应 四、免疫标记的抗原抗体技术 酶联免疫吸附（ELISA）

28 Agglutination/Hemagglutination
Definition - tests that have as their endpoint the agglutination of a particulate antigen Y +  Qualitative agglutination test Ag or Ab

29 Agglutination/Hemagglutination
Quantitative agglutination test Titer Prozone 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 4 Patient 1 2 3 5 6 7

30 Agglutination/Hemagglutination
Definition Qualitative test Quantitative test 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Applications Blood typing Bacterial infections Fourfold rise in titer Practical considerations Easy Semi-quantitative

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35 Immunoelectrophoresis
Method Ags are separated by electrophoresis Ab is placed in trough cut in the agar Ag - Ag Ab + Interpretation Precipitin arc represent individual antigens Ag Ab BACK

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37 Countercurrent electrophoresis
Method Ag and Ab migrate toward each other by electrophoresis Used only when Ag and Ab have opposite charges Ag Ab - + Qualitative Rapid BACK

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39 Complement Fixation Y Methodology Ag No Ag
Ag mixed with test serum to be assayed for Ab Standard amount of complement is added Erythrocytes coated with Abs is added Amount of erythrocyte lysis is determined Ag No Ag Ag Y Patient’s serum Ag Y BACK

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41 酶标记抗体技术 免疫酶技术的基本程序：将酶分子与抗体或抗抗体分子共价结合，酶标记抗体可与存在于组织细胞或吸附于固相载体上的抗原或抗体发生特异性结合。滴加底物溶液后，底物可在酶作用下催化底物水解、氧化或还原等反应，产生有颜色的物质。酶降解底物的量与呈现的颜色成正比。 用于标记的酶：1.辣根过氧化物酶(Horseradish Peroxidase,HRP); 2.碱性磷酸酶(Alkaline phosphatase,AP)。

42 直接法－抗原包被 Ag Y Labeled Ab Tissue Section

43 间 接 法 Ag Y Labeled Anti-Ig Tissue Section Unlabeled Ab

44 夹 心 法－直 接 夹 心 Y Labeled Ab Ag in Patient’s sample Ag Immobilized Solid
夹 心 法－直 接 夹 心 Solid Phase Y Ag Immobilized Ag in Patient’s sample Labeled Ab

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46 直接 法 直接夹心 法

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