Development of a sensitive monoclonalantibody-based enzyme-linked immunosorbent assay for the antimalaria active ingredient artemisinin in the Chinese herb Artemisia annua L.

Abstract

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L. It has been widely used in South-East Asia and Africa as an effective drug against sensitive and multidrug-resistant Plasmodium falciparum malaria. A monoclonal antibody (mAb), designated as 3H2, was generated with artesunate-bovine serum albumin conjugate as the immunogen. mAb 3H2 was used to develop a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) for artemisinin. The concentration of analyte producing 50% of inhibition (IC(50)) and the working range of the icELISA were 1.3 and 0.2-5.8 ng/mL, respectively. The mAb 3H2 recognized the artemisinin analogs artesunate, dihydroartemisinin, and artemether with cross-reactivity of 650%, 57%, and 3%, respectively, but negligibly recognized deoxyartemisinin and the artemisinin precursors arteannuin B and artemisinic acid. The average recoveries of artemisinin fortified in A. annua samples at concentrations from 156 to 5,000 microg/g determined by icELISA ranged from 91% to 98%. The icELISA was applied for the determination of artemisinin in different wild A. annua samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis. The correlation coefficient (R(2)) between the two assays was larger than 0.99, demonstrating a good agreement between the icELISA and HPLC results. This ELISA is suitable for quality assurance of A. annua L. materials.

Validation of ELISA for Quantitation of Artemisinin-Based Antimalarial Drugs

“…The mAb 3H2 has a high sensitivity and low cross-reactivity to the precursors of ART. 31 The optimal concentrations of coating antigen, mAb, and goat anti-mouse IgG-HRP were screened by checkerboard titration. Concentrations of 0.25 mg/mL of coating antigen ATS-ovalbumin (OVA), 0.1 mg/mL of mAb and 0.1 mg/mL of goat anti-mouse IgG-HRP were selected and used throughout this work.…”

“…The icELISA was carried out according to the method previously published. 31 A microtiter plate was first coated with 100 μL of the ATS-OVA conjugate in coating buffer per well for 3 h at 37 C. After three washes with PBST, 50 μL extracts of drugs and 50 μL mAb 3H2 was added to each well for 30 min at 37 C. After three washes with PBST, 100 μL of goat anti-mouse IgG was added to each well and incubated at 37 C for 0.5 h. After the plate was washed with PBST again, 100 μL of substrate solution with OPD and hydrogen peroxide per well was added. The reaction was stopped by adding 50 μL of 2 M H 2 SO 4 .…”

“…26 -30 The instrumentations and methods used to test the contents of ART are usually expensive and time-consuming, and require rigorous sample preparation, whereas isotope-based assays have potential health hazards. Being rapid, cost-effective, sensitive, simple, and convenient, ELISA has become popular for the detection of botanical chemicals and drugs 31 ; we have previously generated a monoclonal antibody (mAb) 3H2 using ATS-bovine serum albumin conjugate as the immunogen. An indirect competitive ELISA (icELISA) was developed to detect ART in the A. annua samples.…”

“…An indirect competitive ELISA (icELISA) was developed to detect ART in the A. annua samples. 31 Here, we have further refined this assay for the quantification of ART and its derivatives. We directly compared the performance of the icELISA with that of the gold standard HPLC method using standards of ART and its derivatives and 22 ART-based antimalarial drugs purchased from the market.…”

Immunochemical Analysis of the Antimalarial Drugs Artemisinin and Artesunate

“…Some research groups have pursued development of immunochemical approaches by designing a convenient indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibodies or monoclonal antibodies for the analysis of AM and its related compounds [21][22][23][24]. Immunoassays are convenient and enable the rapid analysis of multiple samples [25][26][27][28][29][30][31][32][33].…”