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.

Development of monoclonal antibody-based immunoassays for quantification and rapid assessment of dihydroartemisinin contents in antimalarial drugs

“…1A). This poses a major difficulty to develop highly specific mAbs that can distinguish artemisinin and its derivatives, since most methods for conjugating the artemisinin haptens to carrier proteins employed the position 12 in the chemical structures [14, 23, 24]. For example, the artemisinin antibody developed earlier showed 57% and 650% cross-reactivity with DHA and artesunate, respectively [14].…”

Sensitive and rapid lateral-flow assay for early detection of subclinical mammary infection in dairy cows

“…Wells of a polystyrene microtiter plate were coated with 100 µl of OVA conjugated MPO, neutrophil lysate, lymphocyte lysate, lactoperoxidase, lactoferrin and histone (2 μg/ml). After overnight incubation, washing and blocking steps were performed, the monoclonal MPO antibody in various concentration of 1,000, 750, 500, 250, 125, 60 (ng/100 µl) was used, and normal ELISA protocol was followed as described by Zehnacker et al 28 . Finally, the absorbance was taken at 450 nm using a microplate reader (Multiskan Go, Thermo Scientific, Finland).…”