Immunoquantitative analysis of artemisinin from Artemisia annua using polyclonal antibodies

被引:43
作者
Ferreira, JFS
Janick, J
机构
[1] Department of Horticulture, Purdue University, 1165 Horticulture Building, West Lafayette
关键词
Artemisia annua; asteraceae; ELISA; sesquiterpene lactone; artemisinin; HPLC; tissue culture;
D O I
10.1016/0031-9422(95)00542-0
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:97 / 104
页数:8
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