Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the antimalaria active ingredient artemisinin in the Chinese herb Artemisia annua L.
Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the antimalaria active ingredient artemisinin in the Chinese herb Artemisia annua L.
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DOI：
10.1007/s00216-008-2527-5
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发表时间：
2009-02-01
影响因子：
4.3
通讯作者：
Li, Qing X.
中科院分区：
文献类型：
--
作者：
He, Su-Ping;Tan, Gui-Yu;Li, Qing X.
青蒿素是从中草药黄花蒿中分离得到的一种倍半萜内酯，在东南亚和非洲被广泛用于治疗敏感和耐多药恶性疟原虫的有效药物。以青蒿琥酯-牛血清白蛋白结合物为免疫原，制备了一种单抗，命名为3H2。以单抗3H2为底物，建立了一种高灵敏度、高特异性的青蒿素间接竞争酶联免疫吸附试验(IcEL ISA)。该试剂的半数抑制浓度(IC50)为1.3，工作范围为0.2~5.8 ng/mL。单抗3H2识别青蒿素类似物青蒿琥酯、双氢青蒿素和青蒿甲醚，交叉反应率分别为650%、57%和3%，而对脱氧青蒿素和青蒿素前体青蒿素B和青蒿酸的识别作用较弱。青蒿素的平均回收率为91%~98%，浓度范围为156~5,000微克/克。采用离子对夹心法测定了不同野生青蒿素样品中青蒿素的含量，并用高效液相色谱法对测定结果进行了验证。两种方法的相关系数(R-2)均大于0.99，表明ICELSA法与高效液相色谱法测定结果具有较好的一致性。该方法适用于黄花蒿药材的质量控制。
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 (IC50) 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 mu g/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.