Development of assay method of novel metabolites of endogenous prostaglandins and its application

TXB2曾被用作体内TXA2生成的指标，但由于其在采血过程中人为产生，已不能再使用。因此，选择11-脱氢(DH)- txb2，并由研究人员开发了其酶免疫分析法。随后使用市售RIA试剂盒促使我们对11-DH-TXB2的测定方法进行分析，我们意识到提取和纯化是测定前不可避免的步骤。然后，与本课组三共组合作，探索免疫亲和柱提取纯化的简单工艺。免疫亲和柱采用德岛大学山本博士课题组生产的抗11- DH-TXB2单克隆抗体，标准提取方法由课题组车间建立。本课题组各成员分别测定分布已知样品中11-DH-TXB2含量，并应用色谱柱测定病理模型和临床患者中ll-DH- TXB2水平。结果表明，该免疫亲和柱不仅可用于尿液中11-DH-TXB2的测定，也可用于血浆中11-DH-TXB2的测定。然而，将11-DH-TXB2作为TXA2的指标存在诸多困难；动物物种差异的多样性表明，狗很少从TXB2中产生ll-DH- TXB2。而11-DH-TXB2在人体内主要由TXB2产生，其半衰期约为60 min，表明该代谢物可作为临床TXA2生成的指标。此外，我们还发现了许多困难，例如使用开放式的11-DH-TXB2，排除物质的方法，RIA程序中的干扰，同位素标记标准品的可靠性，商业上可获得的（单个批次的含量变化很大）等等。显然，课课组经过三年的研究，建立了11-DH-TXB2的检测方法，包括利用该免疫亲和柱提取和纯化，并揭示了提取过程中的许多困难。少

TXB2 had been used as an index of TXA2 generation in the body, but cannot be used anymore, because of its artifactual generation during blood collection. Instead, 11- dehydro(DH)-TXB2 was chosen and its enzyme immunoassay method was developed by a research member. The subsequent usage of the commercially available RIA kits prompted us to proceed analyses of the determination method of 11-DH-TXB2 and we realized that the extraction and purification was an unavoidable step before assay. Then, the simpler extraction and purification procedure by an immunoaffinity column was explored icolaboration with Sankyo group of this research group. The monoclonal antibody against 11- DH-TXB2, produced by Dr. Yamamoto's group, Tokushima University, was used for this immunoaffinity column and the standard extraction method was established by a workshop of this research group. Each member of this research group measured the 11-DH-TXB2 content in the distributed known samples and the column was applied … More to measure ll-DH- TXB2 levels in pathological models and in clinical patients. It was confirmed that this immunoaffinity columns was useful for assay of 11-DH-TXB2 not only in urine, but also in plasma. There are, however, many difficulties for taking 11-DH-TXB2 as an index of TXA2; the large variety in animal species differences, showing that dogs scarcely produce ll-DH- TXB2 from TXB2. In contrast, 11-DH-TXB2 is produced largely in human from TXB2 and its half life is approximately 60 min, indicating that this metabolite can be taken as an index of TXA2 generation in clinical field. Moreover, we found many difficulties in this assay, such as use of the open type of 11-DH-TXB2, method to exclude substances, disturbed in RIA procedures, reliability of isotope labeled standards, available commercially (large variation of the contentents in individual lots) and others. It is clear that three years studies of this research group established the assay method of 11-DH-TXB2 including extraction and purification using this immunoaffinity column and disclosed many difficulties during the extraction. Less