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Development of immunoaffinity purification and immunoassay of thromboxane and prostacyclin metabolites

血栓素和前列环素代谢物的免疫亲和纯化和免疫分析的发展

基本信息

批准号：
05557013
负责人：
YAMAMOTO Shozo
金额：
$ 4.48万
依托单位：
Tokushima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

在花生四烯酸衍生的生物活性物质中，血栓烷（TX）A_2主要由血小板合成，具有聚集血小板和收缩血管的作用，前列环素主要由血管内皮细胞产生，具有抑制血小板聚集和舒张血管的作用。本研究旨在建立一种简便的免疫亲和纯化方法和一种灵敏的免疫测定方法，以测定11-dehydro-TXB_2、2，3-dinor-TXB_2和2，3-dinor-6-keto-PGF_2，它们<1alpha>的代谢半衰期比TXA_2和6-keto-PGF_2长，是<1alpha>TXA_2和前列环素的稳定降解产物。在第一学年，制备了2，3-二去甲-6-酮-PGF_2的ω-羧基衍生物<1alpha>，并将其ω-羧基与蛋白质结合用于制备抗原。该缀合物用作制备单克隆抗体的抗原，在侧链长度方面具有更高的选择性。用此抗体建立了2，3-二去甲-6-酮-PGF_2的放射免疫分析法和酶免疫分析法<1alpha>。抗体与6-keto-PGF_2的交叉反应性<1alpha>小于0.01%。过氧化物酶联免疫测定法可测定14-1200 pmol范围内的2，3-二去甲-6-酮-PGF_2<1alpha>。其IC_<50>（10）值为120 pmol。第二学年，大量制备了抗2，3-二去甲-6-酮-PGF_<1alpha>2单克隆抗体，并制备了固相柱，用于尿代谢物的免疫亲和层析分离。本方法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。