中国中药杂志

2020, v.45(16) 3900-3907

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高灵敏度黄曲霉毒素B_1单克隆抗体的制备及其在中药材酸枣仁污染快速检测中的应用
Preparation of highly sensitive monoclonal antibody against aflatoxin B_1 and its application in rapid detection of contamination in Ziziphi Spinosae Semen

蒋佳伊;张磊;秦露;骆骄阳;付延伟;秦家安;王长健;欧阳臻;杨美华;
JIANG Jia-yi;ZHANG Lei;QIN Lu;LUO Jiao-yang;FU Yan-wei;QIN Jia-an;WANG Chang-jian;OUYANG Zhen;YANG Mei-hua;School of Pharmacy, Jiangsu University;Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College;

摘要(Abstract):

该研究制备了一种高灵敏度的黄曲霉毒素B_1(aflatoxin B_1,AFB_1)单克隆抗体并基于该抗体建立间接竞争酶联免疫吸附法(indirect competition enzyme-linked immunosorbent assay,ic-ELISA),用于中药材中AFB_1污染的高通量、快速筛查,以保障中药用药安全性。研究中以改进的肟化法修饰AFB_1后,用EDC-NHS法偶联载体蛋白获得AFB_1完全抗原,更加简便且减少环境污染。并利用加大免疫剂量、多种方式注射等对Balb/c雌鼠进行免疫,最终制备得AFB_1单克隆抗体。通过对其免疫特性进行鉴定,结果显示该AFB_1单克隆抗体属于IgG_(2b)型免疫球蛋白,其灵敏度(IC_(50))可达0.15μg·L~(-1),亲和力为2.81×10~8 L·mol~(-1),与AFB_2,AFG_1和AFG_2交叉反应率分别为35.07%,8.75%,1.15%,且与其他类真菌毒素几乎不存在交叉反应。基于该高灵敏度、高特异性抗体建立了ic-ELISA法,并用于中药材酸枣仁样品的检测。采用基质匹配的标准曲线进行计算,AFB_1在0.05~0.58μg·L~(-1)线性良好(R~2=0.992),加样回收率为88.00%~119.0%,检测限为1.69μg·kg~(-1)。采用所建立的方法测定了33批酸枣仁样品中AFB_1的污染情况,其污染水平为3.62~206.58μg·kg~(-1),其检测结果与UHPLC-MS/MS测定结果的线性相关系数为0.996,并且无假阳性和假阴性情况,表明所建立的ic-ELISA准确、可靠,可为中药材酸枣仁中AFB_1污染的快筛提供一种简便、有效的技术手段,同时可为其他中药中AFB_1的检测与监控提供参考。
A highly sensitive monoclonal antibody against aflatoxin B_1(AFB_1) was prepared and an indirect competition enzyme-linked immunosorbent assay(ic-ELISA) was established based on the antibody which was used for high-throughput and rapid screening of AFB_1 contamination in Chinese herbal medicines to ensure the safety of medication. In this study, the structure of AFB_1 was modified by improved oxime method, and the carrier protein was coupled by EDC-NHS method to obtain the complete antigen of AFB_1, which was more convenient and environmental friendly. The Balb/c female mice were immunized using increasing the immunization dose and various ways of injection, and finally the AFB_1 monoclonal antibody was prepared. The AFB_1 monoclonal antibody belongs to IgG_(2 b) immunoglobulin by identifying its immunological characteristics, and its sensitivity(IC_(50)) can reach 0.15 μg·L~(-1), and the affi-nity is 2.81×10~8 L·mol~(-1). The cross-reaction rates of AFB_2, AFG_1, and AFG_2 were 35.07%, 8.75%, and 1.15%, respectively, and there was almost no cross-reactivity with other mycotoxins. Based on the high sensitivity and specificity of the antibody, an ic-ELISA method was established and applied to the determination of AFB_1 contamination in Ziziphi Spinosae Semen. According to the matrix matching standard curve, the linear concentration range for AFB_1 was 0.05-0.58 μg·L~(-1)(R~2=0.992), the recoveries were 88.00%-119.0%, and the detection limit was 1.69 μg·kg~(-1). The AFB_1 in 33 batches of Ziziphi Spinosae Semen samples was determined by ic-ELISA, and the contamination level was 3.62-206.58 μg·kg~(-1). The linear correlation coefficient between the detection results of ic-ELISA and UHPLC-MS/MS was 0.996, and there were no false positive and false negative cases. It indicates that the established ic-ELISA is accurate and reliable, and could provide a simple and effective technique for fast screening of AFB_1 contamination in Ziziphi Spinosae Semen, and also could be considered as the reference for the detection and monitoring of AFB_1 contamination in other Chinese herbal medicines.

关键词(KeyWords): 黄曲霉毒素B_1;单克隆抗体;ic-ELISA;酸枣仁
aflatoxin B_1;monoclonal antibody;ic-ELISA;Ziziphi Spinosae Semen

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81872999);; 中国医学科学院医学与健康科技创新工程项目(2017-I2M-1-013);; 国家重点研发计划项目(2016YFE0112900)

作者(Author): 蒋佳伊;张磊;秦露;骆骄阳;付延伟;秦家安;王长健;欧阳臻;杨美华;
JIANG Jia-yi;ZHANG Lei;QIN Lu;LUO Jiao-yang;FU Yan-wei;QIN Jia-an;WANG Chang-jian;OUYANG Zhen;YANG Mei-hua;School of Pharmacy, Jiangsu University;Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College;

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