Development Of A Colloidal Gold-based Lateral Flow Dipstick Immunoassay Of Artesunate And Dihydroartemisinin And Preliminary Study On Genetic Engineering Antibody Of Brassinolide

Artemisinin-based combination therapy (ACT) plays an indispensable role in malaria control and elimination. However, the circulation of counterfeit, substandard drugs has greatly threatened malaria elimination campaigns. Most methods for the analysis of artemisinin and its derivatives require expensive equipment and sophisticated instrumentation. Yet, a convenient, easy-to-use diagnostic device for rapid evaluation of the quality of artemisinin drugs at the point-of-care is still lacking. Brassinolide (BL) is an essential plant steroid hormone that regulates multiple aspects of growth and development. Applications of genetic engineering antibody in the study of plant hormones physiological activities and their mechanisms have been reported. To modulate the activities of plant hormone, genes of genetic engineering antibody of the plant hormone were inserted into plants for antibody production. But there is no report about the development of brassinolide genetic engineering antibody.Monoclonal antibody (McAb) against artesunate and brassinolide were developed, respectively. And they were further used to develop a colloidal gold-based lateral flow dipstick immunoassay and acquire scFv genes. The main results were as follows.(1) The specific McAb3D82G7against artesunate was prepared. The titer was3.2×10and the mAb showed reactivity with artemisinin (22%), dihydroartemisinin (70%) and, but significantly lower cross reactivity with artemether (0.3%);(2) The specific McAb4D81C11against brassinolide was prepared. The titer was2～4×103and the McAb is IgGl isotype. The icELISA was established based on McAb, the concentration causing50%of inhibition (IC50) and the working range in icELISA for brassinolide was0.9ng mL-1, and0.26ng/mL-3.83ng/mL. The McAb showed no cross reactivity with IAA, GAS, ABA, ZR or iPA. The average recoveries of brassinolide fortified in practical samples ranged from90%to120%.(3) A dipstick assay was developed based on the McAb raised against artesunate. The indicator range of the dipsticks, defined as lowest concentration of the target analytes between which the test line was not visible, were100-200and200-500ng mL-1for artesunate and dihydroartemisinin, respectively. No competitive inhibition was observed up to5,000ng mL-1of quinine, chloroquine diphosphate salt, primaquine phosphate, pyrimethamine, lumefantrine, amodiaquine, piperaquine tetraphosphate tetrahydrate or pyronaridine tetraphosphate. The shelf life of the dipsticks was evaluated under three storage conditions. Storage test showed that the indicator range for artemisinins remained unchanged after a week at37℃and increased four-folds after six months of storage at4℃or ambient temperature.(4) Semi-quantitative analysis of artesunate and dihydroartemisinin in commercial drugs and raw drug materials with the dipsticks produced result agreeable with those determined by high performance liquid chromatography (HPLC).(5) This is the first study about the genetic engineering antibodies against brassinolide. The VH and VL gene of brassinolide monoclonal antibody were cloned from hybridoma cells.