Development of immunoaffinity purification and immunoassay of thromboxane and prostacyclin metabolites

在花生四烯酸衍生的生物活性化合物中，血栓素（TX） A_2在血小板中合成，具有聚集血小板和收缩血管的作用。前列环素主要在血管内皮细胞中产生，可抑制血小板聚集并使血管松弛。人们已经尝试研究这两种生物活性化合物的体内代谢，它们与各种心血管疾病的相关性，并验证它们的药物功效。本课题的目的是建立一种简单的免疫亲和纯化方法和一种灵敏的免疫测定方法，用于测定代谢半衰期比TXB_2和prostaxilin稳定降解产物11-脱氢-TXB_2、2,3-dino -TXB_2和2,3-dino -6-酮- pgf_ < 1α >长的TXB_2和6-酮- pgf_ < 1α >的代谢半衰期。在第一学年，制备了2,3-dinor-6-keto- pgf_ <1alpha>的ω -羧基衍生物，并将其ω -羧基基团结合到蛋白质上制备抗原。该偶联物被用作制备单克隆抗体的抗原，在侧链长度方面具有较高的选择性。利用该抗体建立了2,3-dinor-6-keto- pgf_ <1alpha>的放射免疫分析和酶免疫分析。抗体与6-keto-PGF_<1alpha>的交叉反应性小于0.01%。过氧化物酶联免疫分析法可测定14- 1200pmol范围内2,3-二-6-酮- pgf_ < 1α >。其IC_<50>值为120 pmol。第二学年，大量制备单克隆抗2,3-dinor -6-keto-PGF_<1alpha>抗体，并制备固相柱对尿代谢物进行免疫亲和色谱分离。该技术也适用于关节炎患者滑液中前列腺素E_2和白三烯C_4的测定。

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.