青蒿素类药物特异性单克隆抗体的制备及快速免疫检测方法的建立与应用

发布时间：2018-05-17 22:18

  本文选题：青蒿素 + ACT　； 参考：《中国农业大学》2016年博士论文

【摘要】：青蒿素联合疗法(Artemisinin-based combination therapies, ACTs)是WHO推荐的无并发症恶性疟原虫的一线治疗方案,对于减少全球疟疾引起的致死率和发病率起着重要的作用。但是,在疟疾流行区域劣质的青蒿类药品严重阻碍了疟疾的防控效率,并助涨了疟原虫的抗药性。因此,对于青蒿素类抗疟药的质量监控尤为重要。本研究制备了青蒿素和蒿甲醚的单克隆抗体,用青蒿素和蒿甲醚单克隆抗体研究了免疫学技术(包括酶联免疫检测法以及胶体金免疫检测试纸条)并应用于青蒿素类抗疟药质量检测。主要结果如下：(1)用微生物转化法制备了青蒿素和蒿甲醚的抗原,制备了特异性的蒿甲醚单克隆抗体2G12E1和青蒿素单克隆抗体3H7A10。蒿甲醚单克隆抗体2G12E1与双氢青蒿素、青蒿素、青蒿琥酯、奎宁、磷酸伯氨喹、磷酸氯喹、乙胺嘧啶和本芴醇的交叉反应极低。青蒿素单克隆抗体3H7A10与青蒿琥酯、双氢青蒿素、蒿甲醚当抑制浓度达到20μg mL-1时仍没有交叉反应。(2)用青蒿素单克隆抗体3H7A10建立了青蒿素的ELISA法,IC50为2.6 ng mL-1,检测范围为0.6-11.5 ng mL-1。将青蒿素ELISA应用于野生黄花蒿药材的品质分析和大鼠中的药代动力学研究,与HPLC结果一致。用蒿甲醚单克隆抗体2G12E1建立ELISA方法,IC50为3.7 ng mL-1,检测范围为0.7-19 ng mL-1。用icELISA和HPLC分别对蒿甲醚抗疟药品中的蒿甲醚含量进行了测定,ELISA测定结果与HPLC测定结果有较好的一致性。(3)以蒿甲醚单抗2G12E1为基础,制备了蒿甲醚胶体金免疫检测试纸条,检测范围为500～1000 ng mL-1。以青蒿素单抗3H7A10为基础,制备了青蒿素胶体金免疫检测试纸条,检测范围为250～500 ng mL-1用实验室已制备的特异性青蒿琥酯单抗3D82G6建立了青蒿琥酯的icELISA方法,IC50为6.7 ng mL-1,检测范围为1.6-30.8 ng mL-1。以特异性青蒿琥酯单抗3Dg2G6为基础,制备了青蒿琥酯胶体金免疫检测试纸条,检测范围为1000～2000 ngmL-1。(4)在本实验室中试生产了约2000条蒿甲醚、青蒿琥酯以及双氢青蒿素的胶体金免疫试纸条,并送往疟疾流行区巴布新几内亚、哥伦比亚、印度、赞比亚用于实地检测,检测结果得到确认。从缅甸和肯尼亚收集了164个青蒿素类抗疟药,用试纸条进行了质量分析,检测到一例青蒿琥酯假药。试纸条检测结果与HPLC检测结果完全一致。现又生产了500个青蒿素类药物试纸条分别发往非洲、东南亚等疟疾流行区域进行药品质量快速检测。
[Abstract]:Artemisinin-based combination therapies, ACTs) is a first-line treatment recommended by WHO for Plasmodium falciparum without complications. It plays an important role in reducing the mortality and morbidity caused by malaria worldwide. However, poor quality artemisia drugs in malaria-endemic areas seriously hamper malaria control efficiency and increase the resistance of malaria parasites. Therefore, the quality control of artemisinin antimalarial drugs is particularly important. In this study, monoclonal antibodies against artemisinin and artemether were prepared. The immunological techniques (including enzyme-linked immunosorbent assay (Elisa) and colloidal gold immunoassay strips) were studied with artemisinin and artemisinin monoclonal antibodies and applied to the quality detection of artemisinin antimalarial drugs. The main results are as follows: (1) the artemisinin and artemether antigens were prepared by microbial transformation, and the specific monoclonal antibodies 2G12E1 and 3H7A10 were prepared. The cross reaction of artemisinin monoclonal antibody (2G12E1) with dihydroartemisinin, artesunate, quinine, primary phosphate, chloroquine phosphate, ethylamine pyrimidine and benfluorene was very low. When the inhibitory concentration of artemisinin monoclonal antibody 3H7A10 and artesunate, dihydroartemisinin and artemisinin reached 20 渭 g mL-1, there was no cross reaction. The ELISA assay for artemisinin was established. The IC50 of artemisinin was 2.6 ng mL -1 and the detection range was 0.6-11.5 ng ml -1. The application of artemisinin ELISA to the quality analysis of Artemisia annua and the pharmacokinetic study in rats were in agreement with the results of HPLC. The IC50 was 3.7 ng mL ~ (-1) and the detection range was 0.7-19 ng mL ~ (-1) by ELISA method established by artemether monoclonal antibody 2G12E1. The content of artemether in artemether antimalarial drugs was determined by icELISA and HPLC respectively. The results of Elisa and HPLC were in good agreement. The detection range is 500,000ng mL ~ (-1). Based on artemisinin monoclonal antibody (3H7A10), a gold immunoassay strip of artemisinin colloid was prepared. The detection range was 250,500ng mL-1. The icELISA method for artesunate was established by using the specific artesunate monoclonal antibody (3D82G6) prepared in laboratory. The IC50 of artesunate was 6.7 ng mL -1 and the detection range was 1.6-30.8 ng mL -1. Based on the specific artesunate monoclonal antibody (3Dg2G6), an artesunate colloidal gold immunoassay strip was prepared. The detection range of artesunate colloidal gold immunoassay was 1000ngmL-1.44) about 2000 artesunes were produced in our laboratory. Artesunate and dihydroartemisinin colloidal gold immunized strips were sent to the malaria endemic areas of Papua New Guinea, Colombia, India and Zambia for field testing. The results were confirmed. 164 artemisinin-based antimalarial drugs were collected from Myanmar and Kenya. A case of artesunate was detected by quality analysis with test strip. The results of test strip and HPLC detection are in good agreement. Now 500 artemisinin-based drug test strips have been produced and distributed to Africa, Southeast Asia and other malaria-endemic areas for rapid testing of drug quality.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
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本文编号：1903114