本文選題：青蒿素 + ACT��； 參考：《中國農業(yè)大學》2016年博士論文

【摘要】：青蒿素聯(lián)合療法(Artemisinin-based combination therapies, ACTs)是WHO推薦的無并發(fā)癥惡性瘧原蟲的一線治療方案,對于減少全球瘧疾引起的致死率和發(fā)病率起著重要的作用。但是,在瘧疾流行區(qū)域劣質的青蒿類藥品嚴重阻礙了瘧疾的防控效率,并助漲了瘧原蟲的抗藥性。因此,對于青蒿素類抗瘧藥的質量監(jiān)控尤為重要。本研究制備了青蒿素和蒿甲醚的單克隆抗體,用青蒿素和蒿甲醚單克隆抗體研究了免疫學技術(包括酶聯(lián)免疫檢測法以及膠體金免疫檢測試紙條)并應用于青蒿素類抗瘧藥質量檢測。主要結果如下：(1)用微生物轉化法制備了青蒿素和蒿甲醚的抗原,制備了特異性的蒿甲醚單克隆抗體2G12E1和青蒿素單克隆抗體3H7A10。蒿甲醚單克隆抗體2G12E1與雙氫青蒿素、青蒿素、青蒿琥酯、奎寧、磷酸伯氨喹、磷酸氯喹、乙胺嘧啶和本芴醇的交叉反應極低。青蒿素單克隆抗體3H7A10與青蒿琥酯、雙氫青蒿素、蒿甲醚當抑制濃度達到20μg mL-1時仍沒有交叉反應。(2)用青蒿素單克隆抗體3H7A10建立了青蒿素的ELISA法,IC50為2.6 ng mL-1,檢測范圍為0.6-11.5 ng mL-1。將青蒿素ELISA應用于野生黃花蒿藥材的品質分析和大鼠中的藥代動力學研究,與HPLC結果一致。用蒿甲醚單克隆抗體2G12E1建立ELISA方法,IC50為3.7 ng mL-1,檢測范圍為0.7-19 ng mL-1。用icELISA和HPLC分別對蒿甲醚抗瘧藥品中的蒿甲醚含量進行了測定,ELISA測定結果與HPLC測定結果有較好的一致性。(3)以蒿甲醚單抗2G12E1為基礎,制備了蒿甲醚膠體金免疫檢測試紙條,檢測范圍為500～1000 ng mL-1。以青蒿素單抗3H7A10為基礎,制備了青蒿素膠體金免疫檢測試紙條,檢測范圍為250～500 ng mL-1用實驗室已制備的特異性青蒿琥酯單抗3D82G6建立了青蒿琥酯的icELISA方法,IC50為6.7 ng mL-1,檢測范圍為1.6-30.8 ng mL-1。以特異性青蒿琥酯單抗3Dg2G6為基礎,制備了青蒿琥酯膠體金免疫檢測試紙條,檢測范圍為1000～2000 ngmL-1。(4)在本實驗室中試生產了約2000條蒿甲醚、青蒿琥酯以及雙氫青蒿素的膠體金免疫試紙條,并送往瘧疾流行區(qū)巴布新幾內亞、哥倫比亞、印度、贊比亞用于實地檢測,檢測結果得到確認。從緬甸和肯尼亞收集了164個青蒿素類抗瘧藥,用試紙條進行了質量分析,檢測到一例青蒿琥酯假藥。試紙條檢測結果與HPLC檢測結果完全一致�，F又生產了500個青蒿素類藥物試紙條分別發(fā)往非洲、東南亞等瘧疾流行區(qū)域進行藥品質量快速檢測。
[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.
【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392
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本文編號：1903114