Cyclooxygenasse (COX) is responsible for the oxidation of arachidonic acid to prostaglandin G2 and the subsequent production of PGH2 from PGG2. There are two isoforms of this enzyme, COX-I (a constitutively expressed form) and COX-II (an inducible form). Assay Designs’ human COX-II Enzyme Immunometric Assay (EIA) kit is used for the quantitative measurement of human COX-II.
For the last two years, I have been using the Assay Designs kit for measuring COX-II protein content in the lung cancer cell lysates with and without treatment with the drugs of my interest. This kit contains a monoclonal antibody to human COX-II immobilized on a microtiter plate (provided in the kit) to bind the human COX-II in the standard or samples. A recombinant human COX-II standard is included in the kit. After incubating the standard or the samples for an hour at 37oC, the wells are washed with the washing buffer provided in the kit. A rabbit polyclonal antibody to human COX-II labeled with the enzyme Horseradish peroxidase is then added and incubated at 40ºC. After washing the antibody, substrate solution is added and incubated for 30 mins in the dark. Stop solution is then added and the plate becomes ready to read at 450 nm, preferably with correction between 570 and 590 nm.
This kit is fairly straightforward. The whole process takes about 4 hours including sample preparation and the results among different batches of samples have been sensitive and quite reproducible. This kit uses plates with removable strips, so the number of wells can be used according to the need. Sometimes, dilution of the samples are required depending on the COX-II expression. This kit is compatible with human COX-II samples in cell culture lysates only, it cannot be used for serum or plasma samples due to the low levels of circulating COX-II. This kit is not very cheap to buy. However, if we buy a lot, we get a good discount
In summary, I highly recommend this kit for quantitative measurement of COX-II in cell culture lysates since the results are reliable and the process is very time efficient.