An ELISA method to measure inhibition of the COX enzymes.
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).
Cuendet
Muriel
M
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.
Mesecar
Andrew D
AD
DeWitt
David L
DL
Pezzuto
John M
JM
D000818:Animals; D051545:Cyclooxygenase 1 / Q000302:isolation & purification; D051546:Cyclooxygenase 2 / Q000302:isolation & purification; D016861:Cyclooxygenase Inhibitors / Q000032:analysis*; D015232:Dinoprostone / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D012756:Sheep; D018411:Spodoptera
D016861:Cyclooxygenase Inhibitors; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015232:Dinoprostone