Description
Introduction: Cyclooxygenase (COX) is an enzyme (EC 1.14.99.1) that is responsible for formation of important biological mediators called prostanoids, including prostaglandins, prostacyclin and thromboxane. Pharmacological inhibition of COX can provide relief from the symptoms of inflammation and pain. Non-steroidal anti-inflammatory drugs, such as aspirin and ibuprofen, exert their effects through inhibition of COX. The names "prostaglandin synthase (PHS)" and "prostaglandin endoperoxide synthetase (PES)" are still used to refer to COX. At present, three COX isoenzymes are known: COX-1, COX-2, and COX-3. COX-2 is undetectable in most normal tissues. It is an inducible enzyme, becoming abundant in activated macrophages and other cells at sites of inflammation. Selectivity for COX-2 is the main feature of celecoxib, rofecoxib, and other members of this drug class. Because COX-2 is usually specific to inflamed tissue, there is much less gastric irritation associated with COX-2 inhibitors, with a decreased risk of peptic ulceration. The selectivity of COX-2 does not seem to negate other side-effects of NSAIDs, most notably an increased risk of renal failure, and there is evidence that indicates that there might be an increase in the risk for heart attack, thrombosis, and stroke through a increase in thromboxane. The sale of Rofecoxib (brand name Vioxx) was banned in 2004 because of such concerns. Some other COX-2 selective NSAIDs, such as celecoxib, and etoricoxib, are still on the market.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to COX-2. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for COX-2 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain COX-2, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of COX-2 in the samples is then determined by comparing the O.D. of the samples to the standard curve