Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma

Abstract

Despite significant progress in prevention and therapy, malaria is still one of the world’s leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL⁻¹. This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL⁻¹ and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103 %, with coefficients of variation of <13 %. To date, no immunoassay for DHA has been applied to plasma analysis and this EIA should be very useful in all clinical laboratories for rapid and cost-effective analysis.