Chaper 2(二).ppt

Biochemical Methods The Principle Of ELISA Types of ELISA Sandwich ELISA (双抗体夹心法测抗原,双抗原夹心法测抗体) Indirect ELISA (间接法测抗体) Competitive ELISA (竞争法测抗原/抗体) Sandwich ELISA 工作原理： 利用连接于固相载体上的抗体和酶标抗体分别与样品中被检测抗原分子上两个抗原决定簇结合，形成固相抗体-抗原-酶标抗体免疫复合物。由于反应系统中固相抗体和酶标抗体的量相对于待测抗原是过量的,因此复合物的形成量与待测抗原的含量成正比(在方法可检测范围内)。测定复合物中的酶作用于加入的底物后生成的有色物质量(OD值)，即可确定待测抗原含量。 是检测抗原最常用的ELISA，适用于检测分子中具有至少两个抗原决定簇的多价抗原，而不能用于小分子半抗原的检测。 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。 Indirect ELISA(间接法测抗体) Indirect ELISA Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate. The plate wells or other surface are then coated with serum samples of unknown antibody of interest. 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 以测定抗原为例，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和一定量酶标抗原的混合溶液，使之与固相抗体反应。如受检标本中无抗原，则酶标抗原能顺利地与固相抗体结合。如受检标本中含有抗原，则与酶标抗原以同样的机会与固相抗体结合，竞争性地占去了酶标抗原与固相载体结合的机会，使酶标抗