Development And Application Of Specific Monoclonal Antibodies And Immunoassay Techniques For Artemisinin And Its Derivatives

Artemisinin-based combination therapies (ACTs), as the recommended first-line treatments for uncomplicated Plasmodium falciparum malaria, have played an important role in reducing the global malaria-associated mortality and morbidity. Good-quality ACTs are essential for delivering effective malaria case management, but the increasing proportions of poor-quality (both counterfeit and substandard) drugs in many endemic countries are concerningWe developed monoclonal antibodies (mAb) against artemether and artemisinin, and used them and other artesunate antibody to developed enzyme linked immunosorbent assays and colloidal gold-based lateral flow dipstick immunoassays. The main results were as follows:(1) We used microbial fermentation of artemether and artemisinin to obtain the immunogens, and produced a monoclonal antibody 2G12E1 aganist artemether and a monoclonal antibody 3H7A10 aganist artemisinin.2G12E1 showed low cross reactivities to dihydroartemisinin, artemisinin, artesunate and other major antimalarial drugs.3H7A10 showed no cross reactivities to artesunate, dihydroartemisinin, and artemether for up to 20,000 ng mL-1.(2) An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed, which showed a concentration causing 50% of inhibition (IC50) for artemisinin as 2.6 ng mL-1 and a working range of 0.6-11.5 ng mL-1. The icELISA was applied for the quantification of artemisinin in crude extracts of wild A. annua and the study of pharmacokinetics of artemisinin in rat serum after intraperitoneal injection. The results were highly correlated with those determined by HPLC-UV analysis (R2=0.9919). An icELISA developed by mAb 2G12E1 showed an IC50 for artemether as 3.7 ng mL-1 and a working range of 0.7-19 ng mL-1. The icELISA was applied for determination of artemether content in different commercial drugs and the results were comparable to those determined by HPLC analysis.(3) An artemether dipstick immunoassay was developed by mAb 2G12E1 and the indicator range for artemether was 500-1,000 ng mL-1. A artemisinin dipstick immunoassay was developed by mAb 3H7A10 and the indicator range for artemisinin was 250-500 ng mL-1. We used a mAb 3D82G5 against artesunate which made by our lab before to develop an icELISA. The icELISA showed an IC50 for artesunate as 6.7 ng mL-1 and a working range of 1.6-30.8 ng mL-1. A dipstick immunoassay was developed by mAb 3D82G6 and the indicator range for artesunate was 1,000-2,000 ng mL-1. No interference was observed with artemisinin, dihydroartemisinin, artemether and other commonly used antimalarial drugs for up to 20,000 ng mL-1.(4) We developed about 2000 pieces of artemether and artesunate dipsticks and sent them to different endemic countries including PNG, Columbia, India and Zambia for detecting the artemether and artesunate drugs collected from these places. The artemether, artesunate and dihydroartemisinin dipsticks were used for determination of artemisinins contents in 164 commercial drugs from Myanmar and Kenya, and an artesunate fake drug was found. The results were agreeable with those determined by HPLC. We sent 500 pieces of dipsticks to Africa and Southeast (SE) Asian countries again for field surveys of the drug quality of artemisinin-based antimalarial medicines.