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  3. Enzyme-Linked Immunosorbent Assay

Enzyme-Linked Immunosorbent Assay酶联免疫吸附测定

释义：

Following non-covalent binding of a purified primary ligand to a solid phase, a blocking reagent is added to prevent any non-specific binding. A specific antigen is then allowed to bind to the primary ligand. Unbound antigen is removed by washing and a secondary antibody conjugated to an enzyme (e.g. horseradish peroxidase) is added. Following a washing step to remove unbound secondary ligand, the extent to which a chromogenic substrate (e.g. 3,3', 5,5' tetramethyl benzidine chromogen [TMB]) is converted to a soluble coloured product by the conjugated enzyme in a given time is determined by spectrophotometry using a standard microplate absorbance reader. A similar type of approach can be utilized to detect enzymatic activities. The substrate, attached to a solid phase is incubated in the presence of the enzyme and the enzymatic modification is monitored by an antibody that is specific for the modified substrate (for instance a phosphorylated protein).

通过非共价结合纯化的主要配体到固相后，添加阻断试剂以防止任何非特异性结合。然后，允许特定抗原与主要配体结合。未结合的抗原通过洗涤去除，并添加与酶结合的二级抗体 (例如辣根过氧化物酶)。 洗涤步骤后去除未结合的二级配体，通过光谱光度法使用标准微孔板吸光度计来确定色谱底物 (例如 3,3'，5,5' 四甲基苯胺色谱底物 [TMB]) 在给定时间内转化为可溶的有色产物的程度。类似的方法也可用于检测酶活性。将底物固定到固相上，与酶共同孵育，并通过特定于修饰底物的抗体来监测酶修饰 (例如磷酸化蛋白质)。

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