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

Abstract

BackgroundSince 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).ResultsAnti-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 %.ConclusionsThe 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.

Assessment of quantitative and semi-quantitative biological test methods of artesunatein vitro

“…Developing a method for artesunate quantification has multiple shortcomings because the drug is poorly soluble and readily hydrolyses in dihydroartemisinin (DHA) in aqueous acidic and neutral media [1,11]. Several previous attempts have been made to develop simple quantitative methods for artesunate dosage including immunoassay-based tests (LFIA, ELISA) [15,17,21]. While some of them showed good performances, they are still difficult to implement in resource-limited countries.…”

“…Methods for artesunate quantification including UV-VIS spectrophotometric [13,23], high performance liquid chromatography (HPLC) [22], and liquid chromatography with mass spectrometry detection (LC-MS or LC-MS/MS) [8,26] were previously described, but they are costly and difficult to implement in malaria endemic regions. Whereas attempts were made to develop low cost methods such as lateral flow immunoassay (LFIA) [15] and enzyme-linked immunosorbent assay (ELISA) [17], they are still difficult to implement in resource-limited countries. In this context, a bioassay based on antimalarial activity measurement appears to be a suitable method for artesunate quantification in various samples.…”

Preparation of Luciferase-fused Peptides for Immunoassay of Amyloid Beta

“…7 Conjugation of HRP to antibodies or antigens is performed by organic chemical methods using condensation reagents rather than genetic engineering methods. 8,9 Use of the former technique often causes a decrease in the interaction ability of the antibody because of an unfavorable modification of epitopes and difficulty in preparing conjugates with a uniform structure. 10 Gene engineering is not generally used for the preparation of a fused protein with a uniform structure, primarily because of difficulty in the expression of HRP by a protein expression system using host cells, such as Escherichia coli.…”