Development of an indirect competitive enzyme-linked immunosorbent assay (icELISA) using highly specific monoclonal antibody against paclitaxel
Development of an indirect competitive enzyme-linked immunosorbent assay (icELISA) using highly specific monoclonal antibody against paclitaxel
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DOI：
10.1007/s11418-012-0708-1
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发表时间：
2013-07-01
影响因子：
3.3
通讯作者：
Morimoto, Satoshi
中科院分区：
文献类型：
--
作者：
Chao, Zhi;Tan, Mingming;Morimoto, Satoshi
紫杉醇是红豆杉的主要活性成分，是一种重要的抗癌药物。为制备紫杉醇单克隆抗体用于免疫分析，将7-木糖基紫杉醇（7-xyltaxol）与载体蛋白牛血清白蛋白（BSA）偶联，构建免疫原，采用基质辅助激光解吸电离飞行时间质谱法测定XylTax-BSA偶联物中半抗原比例。用该缀合物免疫小鼠后，通过将脾细胞与小鼠骨髓瘤细胞系SP2/0融合获得分泌抗紫杉醇的MAb的杂交瘤。经杂交瘤筛选，获得了抗紫杉醇单克隆抗体3A 3，其对紫杉醇具有较高的特异性（与其它天然紫杉烷类化合物的交叉反应性：7-木糖基紫杉醇，31.8%;三尖杉宁碱，6.17%;浆果赤霉素III、10-脱乙酰基浆果赤霉素III、1-羟基浆果赤霉素I、13-乙酰基-9-二氢浆果赤霉素III和1-乙酰氧基-5-脱乙酰基浆果赤霉素I，<0.11%）。利用单克隆抗体3A 3建立了紫杉醇的间接竞争酶联免疫吸附试验（icELISA），检测范围为0.098-312.5 μ g/ml。测定紫杉醇含量在各种紫杉树样品与此icELISA导致回收率范围从92%至94.8%，内和批间变异分别为3.6%和4.7%。这种icELISA为各种目的的紫杉醇测定提供了一种有价值的方法。
Paclitaxel, the major active component of the yew tree, is used as an important anti-cancer agent. To obtain the monoclonal antibody (MAb) against paclitaxel for paclitaxel determination using immunoassay, 7-xylosyltaxol was conjugated to the carrier protein bovine serum albumin (BSA) to construct the immunogen, and the ratio of hapten in XylTax-BSA conjugate was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. After immunization of mice with this conjugate, hybridomas secreting MAbs against paclitaxel were obtained by fusing the splenocytes with the mouse myeloma cell line SP2/0. After hybridoma screening, the anti-paclitaxel MAb 3A3 was obtained, which showed a relatively high specificity to paclitaxel (cross-reactivities against other naturally occurred taxanes: 7-xylosyltaxol, 31.8 %; cephalomannine, 6.17 %; baccatin III, 10-deacetyl-baccatin III, 1-hydroxybaccatin I, 13-acetyl-9-dihydrobaccatin III and 1-acetoxyl-5-deacetyl-baccatin I, < 0.11 %). Using the MAb 3A3, we established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for paclitaxel determination with a detection range of 0.098-312.5 mu g ml(-1). Determination of paclitaxel contents in various yew tree samples with this icELISA resulted in recovery rates ranging from 92 to 94.8 %, and intra- and inter-assay variations of 3.6 and 4.7 %, respectively. This icELISA provides a valuable method of paclitaxel determination for various purposes.