Enzyme-Linked Immunosorbent Assays.
Enzyme-Linked Immunosorbent Assays.
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DOI：
10.1002/0471142735.im0201s110
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发表时间：
2015-08-03
影响因子：
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通讯作者：
Hornbeck, Peter V
中科院分区：
文献类型：
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作者：
Hornbeck, Peter V
本单元描述了六种不同的ELISA系统，用于检测特异性抗体、可溶性抗原或细胞表面抗原。在所有六种体系中，可溶性反应物在特异性结合到固相反应物后从溶液中除去。在前四个方案中，固相反应物通过将抗原或抗体吸附到塑料微量滴定板上来制备;在接下来的两个方案中，固相反应物是细胞相关分子。在所有方案中，固相试剂与共价偶联至酶的二级或三级反应物一起孵育。洗去未结合的缀合物，并加入显色或荧光底物。当底物被结合的酶缀合物水解时，产生有色或荧光产物。最后，目视或用微量滴定板读数器检测产品。生成的产物量与供试混合物中分析物的量成比例。其中一种支持方案可用于优化不同的ELISA。第二个支持方案提供了一种制备碱性磷酸酶缀合物的方法。
This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. One of the support protocols can be used to optimize the different ELISAs. A second support protocol presents a method for preparing alkaline phosphatase conjugates.