Preparation Of Single-Chain Variable Fragments And Development Of Immunoassays Techniques For Artemether And Dihydroartemisinin

Artemether and dihydroartemisinin are derivatives of artemisinin and are recommended by the World Health Organization as first-line durgs for the treatment of falciparum malaria.However,in malaria endemic areas,counterfeit and shoddy artemisinin drugs have seriously affected the efficiency of malaria prevention and control.In recent years,antibody-based enzyme-linked immunosorbent assay（ELISA）has been gradually applied to the quality monitoring of antimalarial drugs.The development of new antibody technologies has provided the conditions for the establishment of more efficient and green immunoassays.Based on the previous research on monoclonal antibodies,this thesis has prepared single-chain antibodies and immunoassays for artemether and dihydroartemisinin respectively,and successfully applied them to the quality analysis of artemisinin-based antimalarials.In this study,the gene sequences of the light and heavy chain variable regions of artemether and dihydroartemisinin antibodies were cloned and determined.prokaryotic and eukaryotic expression vectors were constructed to achieve soluble expression of single-chain antibodies to artemether and dihydroartemisinin,respectively.The scFv was purified by affinity chromatography and the activity of scFv was evaluated using Surface Plasmon Resonance（SPR）and ELISA.The ELISA method was established for the detection of artemether and dihydroartemisinin.The results are as follows:（1）Based on the Artemether monoclonal antibody 2G12E1,the gene of the variable region of artemether monoclonal antibody was cloned and determined,and spliced into scFv.The p MAL-p5x-scFv prokaryotic expression vector was constructed for the E.coli inducible expression of Artemether scFv.The affinity of scFv to antigen was 1.90×10^-8 M by SPR.Based on the scFv,the ic ELISA method of artemether was established,the IC₅₀ value and the working range based on 20%-80%inhibition were 4.33 ng/m L,and 1.05-22.65 ng/m L.The scFv showed low cross-reactivity with artemisinin（1.47%）,dihydroartemisinin（0.75%）,and artesunate（<0.03%）.The ic ELISA method determined the active ingredients of artemether antimalarial drugs and APIs,and the results showed good consistency with Ultra performance liquid chromatography（UPLC）.（2）The pB-CAG-puro-T-sfGFP-artemisinin scFv eukaryotic expression vector was constructed for soluble expression in human embryonic kidney 293T cells（HEK239T）.Based on this scFv,the ic ELISA method was established,the IC₅₀ value and working range were3.54 ng/m L and 0.78-17.60 ng/m L.The scFv showed low cross-reactivity with artemisinin（1.48%）,dihydroartemisinin（0.55%）and artesunate（<0.02%）.（3）The heavy and light chain variable region genes were cloned and determined using hybridoma cells secreting the dihydroartemisinin cloned antibody 2G₁1G₄ as material.It was constructed by ligation as a dihydroartemisinin-scFv single chain antibody gene,and after ligation with the expression vector p MAL-p5x,dihydroartemisinin scFv was expressed in E.coli supernatant fused with the MBP tag.The affinity of scFv to antigen was determined by SPR to be 6.42×10^-8 M.The ic ELISA method was established with the IC₅₀ value and working range were 2.15 ng/m L and 0.42-13.50 ng/m L.The scFv showed low cross-reactivity with artemisinin（46.74%）,and artesunate（1.21%）,artemether（0.40%）.Determination of the active ingredients of dihydroartemisinin antimalarial drugs and APIs using ic ELISA showed good agreement with UPLC.（4）The pB-CAG-puro-T-sfGFP-dihydroartemisinin scFv eukaryotic expression vector was constructed and soluble expression was performed in HEK293T cells.Based on this scFv,ic ELISA was established with IC₅₀ of 4.95 ng/m L,working range of 0.88-27.33 ng/m L.The scFv showed low cross-reactivity with artemisinin（26.81%）,artesunate（2.40%）and artemether（0.29%）.In summary,scFv of artemether and dihydroartemisinin was successfully prepared by prokaryotic and eukaryotic expression systems,and the highly sensitive ELISA analysis method was established and applied to the detection of artemether and dihydroartemisinin drugs.It provides novel antibody materials for the development of rapid assays for artemether and dihydroartemisinin,and lays the foundation for subsequent studies on the molecular recognition mechanism of antigen-antibodies to artemisinin-based drugs and the in vitro evolution of antibodies.