Oiso Shigeru, Morinaga Osamu, Goroku Takaaki, Uto Takuhiro, Shoyama Yukihiro, Kariyazono Hiroko
*Graduate School of Pharmaceutical Sciences; Departments of †Pharmaceutical Health Care Sciences; and ‡Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, Japan.
Ther Drug Monit. 2015 Oct;37(5):594-9. doi: 10.1097/FTD.0000000000000184.
Dabigatran (DT) is a direct thrombin inhibitor used to prevent venous and arterial thromboembolism due to atrial fibrillation. DT is the active form of the commercially available prodrug DT etexilate. Although DT has many clinical advantages over warfarin, it increases the incidence of bleeding in patients with renal dysfunction. Circulating levels of DT are increased in such patients because it is mainly eliminated by renal excretion. Therapeutic drug monitoring may therefore help to prevent adverse DT effects, but no method for measuring circulating DT levels has been reported, except for an analysis by liquid chromatography-tandem mass spectrometry. This study sought to develop a novel enzyme-linked immunosorbent assay (ELISA) to measure DT concentrations.
Mice were immunized with a DT-keyhole limpet hemocyanin conjugate to generate an anti-DT antibody. Immunized mouse splenocytes and myeloma cells (SP2/0) were fused to obtain an anti-DT monoclonal antibody (DT-mAb). DT-mAb and DT solutions were added to microplate wells coated with a DT-human serum albumin conjugate. DT concentrations were determined based on the principles of ELISA.
DT-mAb was successfully purified from a hybridoma, and the competitive ELISA developed using this DT-mAb could evaluate DT concentrations ranging from 7.8 to 125 ng/mL. The ELISA signal was not linear using DT-spiked serum; however, it was linear when serum ultrafiltrate was used. Weak cross-reactivity with DT etexilate was detected, but no cross-reactivity was observed with other structurally related drugs or drugs commonly used for the treatment of atrial fibrillation.
The developed competitive ELISA is a valuable and specific tool to analyze free DT in serum ultrafiltrate for therapeutic drug monitoring and pharmacokinetic studies.
达比加群（DT）是一种直接凝血酶抑制剂，用于预防心房颤动引起的静脉和动脉血栓栓塞。DT是市售前体药物达比加群酯的活性形式。尽管DT相对于华法林具有许多临床优势，但它会增加肾功能不全患者出血的发生率。此类患者中DT的循环水平会升高，因为它主要通过肾脏排泄消除。因此，治疗药物监测可能有助于预防DT的不良反应，但除了液相色谱 - 串联质谱分析外，尚未报道过测量循环DT水平的方法。本研究旨在开发一种新型酶联免疫吸附测定法（ELISA）来测量DT浓度。
用DT - 钥孔戚血蓝蛋白偶联物免疫小鼠以产生抗DT抗体。将免疫的小鼠脾细胞与骨髓瘤细胞（SP2/0）融合以获得抗DT单克隆抗体（DT - mAb）。将DT - mAb和DT溶液加入包被有DT - 人血清白蛋白偶联物的微孔板孔中。根据ELISA原理测定DT浓度。
成功从杂交瘤中纯化出DT - mAb，使用该DT - mAb开发的竞争性ELISA可评估7.8至125 ng/mL范围内的DT浓度。使用添加DT的血清时，ELISA信号不是线性的；然而，使用血清超滤物时它是线性的。检测到与达比加群酯有弱交叉反应，但未观察到与其他结构相关药物或常用于治疗心房颤动的药物有交叉反应。
所开发的竞争性ELISA是一种有价值且特异的工具，可用于分析血清超滤物中的游离DT，以进行治疗药物监测和药代动力学研究。
