Development of immunoaffinity purification and immunoassay of thromboxane and prostacyclin metabolites

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Among the bioactive compounds derived from arachidonic acid, thromboxane (TX) A_2, which is synthesized in platelets, aggregates platelets and constricts blood vessels.Prostacyclin, which is produced predominantly in vascular endothelial cells, inhibits platelet aggregation and relaxs blood vessels. Ateempts have been made for investigation of the in vivo metabolism of these two bioactive compounds, their relevance to various cardiovascular diseases, and validation of their pharmaceutical efficacies.The purposes of this research project are the developments of a simple immunoaffinity purification method and a sensitive immunoassay for 11-dehydro-TXB_2,2,3-dinor-TXB_2 and 2,3-dinor-6-keto-PGF_<1alpha> with metabolic half lives longer than TXB_2 and 6-keto-PGF_<1alpha> which are stable degradation products of very labile TXA_2 and prostacyclin. In the first academic year, an omega-carboxy derivative of 2,3-dinor-6-keto-PGF_<1alpha> was prepared, and its omega-carboxy group was bound to a protein for preparation of an antigen. The conjugate was used as an antigen for preparation of a monoclonal antibody with a higher selectivity in terms of the sidechain length. By the use of this antibody we set up a radioimmunoassay and an enzyme immunoassay of 2,3-dinor-6-keto-PGF_<1alpha>. Cross-reactivity of the antibody with 6-keto-PGF_<1alpha> was less than 0.01%. The peroxidase-linked immunoassay allowed the determination of 2,3-dinor-6-keto-PGF_<1alpha> in a range of 14-1200 pmol. Its IC_<50> value was 120 pmol. In the second academic year, the monoclonal anti-2,3-dinor -6-keto-PGF_<1alpha> antibody was prepared in a large amount, and a solid phase column was prepared for immunoaffinitychromatographic isolation of the urinary metabolite. This technique was also applied to prostaglandin E_2 and leukotriene C_4 contained in synovial fluid of arthritis patients.