title: Immunoquantitative analysis of artemisinin from Artemisia annua using polyclonal antibodies.
abstract: Artemisinin was derivatized to dihydroartemisinin carboxymethylether in three steps, without disturbing the peroxide bridge, and then linked to either thyroglobulin (TGB) or bovine serum albumin (BSA). The artemisinin-TGB and -BSA conjugates were injected in female New Zealand rabbits but only the artemisinin-TGB conjugate generated polyclonal antibodies. An enzyme-linked immunosorbent assay (ELISA) was developed and the specificity of the antibodies was confirmed by comparison with pre-immune serum and by competitive assays using different dilutions of artemisinin standards. Although anti-artemisinin antibodies cross-reacted with artemisitene and dihydroartemisinin at all dilutions used, cross-reaction with deoxyartemisinin, artemisinic acid, and arteannuin B occurred only at high concentrations. ELISA successfully detected artemisinin from crude extracts in concentrations as low as 1.5 ng ml-1; and was epsilon 400-fold more sensitive than the HPLC-EC. The ELISA successfully detected and quantified artemisinin in different organs of greenhouse-grown plants and in eight clones of Artemisia annua grown in tissue culture but artemisinin was overestimated owing to cross-reactivity of the antibodies with artemisinin-related compounds present in the samples. Despite overestimation of artemisinin content, the correlations between ELISA and HPLC-EC were r = 0.92 when samples were diluted 100 times, and r = 0.90 when samples were diluted 500 times, indicating that ELISA is a potential tool for screening large A. annua populations.
authors.affiliation: Department of Horticulture, Purdue University, West Lafayette, IN 47907-1165, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Ferreira
authors.forename: J
authors.initials: J
authors.lastname: Janick
mesh_terms: D000818:Animals; D000906:Antibodies; D000962:Antimalarials / Q000032:analysis*; D037621:Artemisinins; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D010946:Plants, Medicinal; D011817:Rabbits / Q000276:immunology; D012680:Sensitivity and Specificity; D012717:Sesquiterpenes / Q000032:analysis*
chemical_list: D000906:Antibodies; D000962:Antimalarials; D037621:Artemisinins; D012717:Sesquiterpenes; C031327:artemisinin