一种用于测量环氧化酶（COX）酶抑制作用的酶联免疫吸附测定（ELISA）方法。
An ELISA method to measure inhibition of the COX enzymes.
作者信息
Cuendet Muriel, Mesecar Andrew D, DeWitt David L, Pezzuto John M
机构信息
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, College of Pharmacy, Nursing and Health Sciences, and The Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA.
出版信息
Nat Protoc. 2006;1(4):1915-21. doi: 10.1038/nprot.2006.308.
The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. The first part of the assay consists of incubating arachidonic acid, cyclooxygenase and the test samples to generate prostaglandins. The second part uses an ELISA method to quantify the amount of PGE2 produced by the enzymatic reaction. The isolation of COX-1 and COX-2 enzymes is also described. This protocol can be completed in approximately 23 h, including 16-h and 4-h incubation phases. This does not include enzyme preparation (3 h for COX-1 and 24 h for COX-2) or preparation of ELISA plates (23 h, including incubation).
环氧化酶（COX）反应可通过测量耗氧量、过氧化物酶共底物氧化或前列腺素（PG）检测来监测。本方案描述了一种在有或没有潜在抑制剂存在的情况下，通过定量花生四烯酸酶促转化产生的前列腺素E2（PGE2）来测量环氧化酶活性的方法。这种高通量方法的优点是它直接测量环氧化酶活性且所需酶量很少。测定的第一部分包括将花生四烯酸、环氧化酶和测试样品孵育以生成前列腺素。第二部分使用酶联免疫吸附测定（ELISA）方法来定量酶促反应产生的PGE2的量。还描述了COX-1和COX-2酶的分离方法。本方案大约可在23小时内完成，包括16小时和4小时的孵育阶段。这不包括酶制备（COX-1为3小时，COX-2为24小时）或ELISA板制备（23小时，包括孵育）。
Zotero 插件