biochemical methods chapter 2general identification methods 二 生物检测常用鉴别方法二.pptx

Biochemical Methods;Section 4: Protein Detection Tech 蛋白质检测技术;Antigen is immobilized on a solid support (usually a polystyrene (聚苯乙烯) microtiter plate) . After the antigen is immobilized, the antibody sample is added, forming a complex with the antigen,which can be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent(清洁剂) solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antibody in the sample. ;The Principle Of ELISA; Immunosorbent: 固相的抗原或抗体，即“免疫吸附剂” Conjugate: 酶标记的抗原或抗体，即“酶联物” Substrate: 酶反应的底物 ;Types of ELISA;Sandwich ELISA;操作步骤： （1）将特异性抗体与固相载体联结，形成固相抗体。洗涤除去未结合的抗体及杂质。 （2）加受检标本，保温反应。标本中的抗原与固相抗体结合，形成固相抗原抗体复合物。洗涤除去其他未结合物质。 （3）加酶标抗体，保温反应。固相免疫复合物上的抗原与酶标抗体结合。彻底洗涤未结合的酶标抗体。此时固相载体上带有的酶量与标本中受检抗原的量相关。 （4）加底物显色。固相上的酶催化底物成为有色产物。通过比色，测知标本中抗原的量。;A sandwich ELISA Procedure: (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody (enzyme-linked antibody) is added, and binds to antigen; (4) substrate is added, and is converted by enzyme to detectable form. ;Indirect ELISA(间接法测抗体);Indirect ELISA ; Secondary antibodies, which will bind to unknown antibody of interest, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. Wash the plate, so that excess unbound enzyme-antibody conjugates are removed. Apply a substrate which is converted by the enzyme to elicit(引起) a chromogenic(发色的) or fluorogenic or electrochemical signal. View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device. ;Competitive ELISA竞争法测抗原/抗体; 以测定抗??为例，受检抗原和酶标抗原竞争与固相抗体结合，因此结合于固相的酶标抗原量与受检抗原的量呈反比。操作步骤如下： （1）将特异抗体与固相载体连接，形成固相抗体。洗涤。 （2）待测管中加受检标本