第十二章免疫重点分析.ppt

第十二章 免疫学实验技术及其应用 山东师范大学生命科学学院 杨桂文 （五）Western Blotting FIGURE 6-12 In Western blotting, a protein mixture is (a) treated with SDS, a strong denaturing detergent, (b) then separated by electrophores is an SDS polyacrylamide gel (SDS) which separates the components according to their molecular weight; lower molecular weight components migrate farther than higher molecular weight ones. (c) 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. (d) Addition of enzyme-linked antibodies detects the antigen of interest, and (e) 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. Alternatively, a chemiluminescent ELISA can be used to generate light that is readily detected by exposure of the blot to a piece of photographic film. （六）Flow Cytometry and Fluorescence 第二节 抗体的分离提纯 亲和层析 * * 第一节 基于抗原—抗体反应的免疫学实验技术 （一）高度特异性 （二）表面可逆结合 （三）适当的浓度比例和带现象 （四）特异性结合与反应可见两个阶段 一、抗原—抗体反应的一般规律和特点 FIGURE 6-1 The interaction between an antibody and an antigen depends on four types of noncovalent forces: (1) hydrogen bonds, in which a hydrogen atom is shared between two electronegative atoms; (2) ionic bonds between oppositely charged residues; (3) hydrophobic interactions, in which water forces hydrophobic groups together; and (4) van der Waals interactions between the outer electron clouds of two or more atoms. In an aqueous environment, noncovalent interactions are extremely weak and depend upon close complementarity of the shapes of antibody and antigen. FIGURE 6-4 Precipitation reactions. (a) Polyclonal antibodies can form lattices, or large aggregates, that precipitate out of solution. However, if each antigen molecule contains only a single epitope recognized by a given monoclonal antibody, the antibody can link only two mole