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 Zhenjiang 212013, China 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 Beijing 100193, China.
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 Beijing 100193, China.
Zhongguo Zhong Yao Za Zhi. 2020 Aug;45(16):3900-3907. doi: 10.19540/j.cnki.cjcmm.20200522.201.
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 2.81×108 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)(R2=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.
制备了一种针对黄曲霉毒素B_1（AFB_1）的高灵敏度单克隆抗体，并基于该抗体建立了间接竞争酶联免疫吸附测定法（ic-ELISA），用于高通量快速筛查中药材中AFB_1污染情况，以确保用药安全。本研究采用改进肟化法修饰AFB_1结构，通过EDC-NHS法偶联载体蛋白，获得AFB_1完全抗原，该方法更简便且环保。采用递增免疫剂量及多种注射方式免疫Balb/c雌性小鼠，最终制备出AFB_1单克隆抗体。通过鉴定其免疫学特性，该AFB_1单克隆抗体属于IgG_(2 b)免疫球蛋白，其灵敏度（IC_(50)）可达0.15 μg·L(-1)，亲和力为2.81×108 L·mol(-1)。AFB_2、AFG_1和AFG_2的交叉反应率分别为35.07%、8.75%和1.15%，与其他霉菌毒素几乎无交叉反应。基于该抗体的高灵敏度和特异性，建立了ic-ELISA方法并应用于酸枣仁中AFB_1污染的测定。根据基质匹配标准曲线，AFB_1的线性浓度范围为0.05 - 0.58 μg·L(-1)（R2 = 0.992），回收率为88.00% - 119.0%，检测限为1.69 μg·kg(-1)。采用ic-ELISA法测定了33批次酸枣仁样品中的AFB_1，污染水平为3.62 - 206.58 μg·kg~(-1)。ic-ELISA检测结果与超高效液相色谱-质谱联用（UHPLC-MS/MS）法的线性相关系数为0.996，无假阳性和假阴性情况。表明所建立的ic-ELISA准确可靠，并可为快速筛查酸枣仁中AFB_1污染提供一种简单有效的技术，也可为其他中药材中AFB_1污染的检测与监测提供参考。
