Cyclooxygenase assays

In issue N° 16 of SPIbio Communication, we discussed the two identified isoforms of cyclooxygenase (COX), an enzyme which is essential for the metabolism of arachidonic acid to thromboxane and prostaglandins. The same issue also touched upon some examples of the use of polyclonal and monoclonal antibodies developed by C.E.A., notably for the fine detection of two enzymes during immunoblotting experiments. It very rapidly became apparent that, in order to improve our understanding of cyclooxygenases and study them more effectively, recourse was necessary to methods of measuring these enzymes either directly or via their enzymatic activity. For this reason C.E.A. developed methods of determination by immunometric assay at two specific sites on each of the two isoforms, COX-1 and COX-2.

In the case of COX-1 (constitutive form), the microtitration plates are coated with polyclonal antibodies corresponding to the total immunoglobulin fraction of an antiserum from a rabbit immunised with COX-1 obtained from a ram. The enzyme tracer used is a covalent conjugate of a murine monoclonal antibody and acetylcholinesterase. The standard used in this assay corresponds to COX-1 purified from seminal vesicles of the ram. Under these conditions we observed a limit of detection for the assay of about 100 pg/ml and good parallel with serial dilutions of human samples.

In the case of COX-2 (inducible form), the plates are coated by affinity with purified rabbit polyclonal antibodies, and directed against the sequence (570-581) of human COX-2 (different sequence from that of human COX-1). The enzyme tracer corresponds to a murine monoclonal antibody directed against the sequence (580-598) of human COX-2 (a sequence absent from human COX-1) labelled with acetylcholinesterase. The standard used in this system is a peptide corresponding to the sequence (570-595) of human COX-2.

This assay enables the limit of detection to be lowered to about 500 fmol/ml and gives good parallel with serial dilutions of human samples. These two assays have proved highly efficient for evaluating the level of expression of the two isoforms in stimulated human endothelial cells, and showed correlation to the results obtained by immunoblotting. They are sufficiently sensitive to yield important information regarding the regulation of each of these two enzymes in biological or pathological situations, and facilitate the handling of large numbers of samples. Two problems have yet to be resolved, however: the extraction yield of these (membrane) enzymes by the detergent used (CHAPS), which is still unknown, and the lack of standards (purified or recombinant human proteins) suitable for obtaining exact values.

Another solution involves the study of the enzymatic activity of these molecules by measuring the metabolic products of arachidonic acid such as PGE2 and 6-keto PGF1alpha. Thus an in vitro study revealed that the enzymatic activity of COX-2 measured via the production of PGE2 was inhibited in the presence of a new anti-inflammatory agent, NS-398, whereas that of COX-1 was unchanged (see references below).

If you are interested in the above-mentioned studies, we shall be happy to discuss with you the pro-tocols and assays that could be used for your molecules.

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