Development of a simple and specific direct competitive ELISA for the determination of artesunate using an anti-artesunate polyclonal antiserum

Background: Since artesunate (ART) became a vital component of artemisinin (ARM)-based combination therapies for the treatment for malaria,counterfeit ART drugs have spread in regions of Southeast Asia and Africa. The consumption of counterfeit ART drugs has resulted in the death of many patients. Thus,evaluating the quality of ART drugs is needed. There are several methods for quantitating the ART content in tablets,the most common being a high-performance liquid chromatography. However,that method is hampered by the need for expensive equipment and a rather time-consuming process of extraction. By contrast,enzyme-linked immunosorbent assays (ELISAs) are faster and much less expensive,and they require less sample preparation than the above method. The objective of the present study was to establish a simple and specific direct competitive ELISA for the determination of ART concentrations using an anti-ART polyclonal antibody (pAb). Results: Anti-ART pAb was raised in mice,and ART-horseradish peroxidase (HRP) conjugate was produced. A direct competitive ELISA was performed by simultaneously incubating ART and the ART-HRP conjugate with the anti-ART pAb over a second antibody. Subsequently,the enzyme activity of the remaining ART-HRP conjugate was measured. The intra-and inter-assay coefficients of variation of the ELISA were less than 10 % in the range of 0.3 to 30 ng/ml with a detection limit of 0.1 ng/ml. The cross-reactivities of the anti-ART pAb with ARM and dihydroartemisinin were 0.12 and 0.04 %,respectively,and those with other antimalarial drugs were negligible. Furthermore,the recovery of 10 or 50 ng/ml ART added to the drug tablet solutions containing an expected amount of 10 ng/ml was estimated by the ELISA. The recovery of the ART amount ranged between 98 and 106 %,with coefficient variations of less than 7.0 %. Conclusions: The present ELISA is a simple and specific method for the determination of ART concentrations. Thus,this ELISA can be used to identify ART counterfeits and substandard drugs and to quantify the ART drugs