Preparation of highly sensitive monoclonal antibody against aflatoxin B_1 and its application in rapid detection of contamination in Ziziphi Spinosae Semen.
10.19540/j.cnki.cjcmm.20200522.201
- Author:
Jia-Yi JIANG
1
;
Lei ZHANG
2
;
Lu QIN
2
;
Jiao-Yang LUO
2
;
Yan-Wei FU
2
;
Jia-An QIN
2
;
Chang-Jian WANG
2
;
Zhen OUYANG
3
;
Mei-Hua YANG
2
Author Information
1. 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.
2. 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.
3. School of Pharmacy, Jiangsu University Zhenjiang 212013, China.
- Publication Type:Journal Article
- Keywords:
Ziziphi Spinosae Semen;
aflatoxin B_1;
ic-ELISA;
monoclonal antibody
- MeSH:
Aflatoxin B1;
analysis;
Animals;
Antibodies, Monoclonal;
Drug Contamination;
Enzyme-Linked Immunosorbent Assay;
Female;
Mice;
Semen;
chemistry;
Tandem Mass Spectrometry
- From:
China Journal of Chinese Materia Medica
2020;45(16):3900-3907
- CountryChina
- Language:Chinese
-
Abstract:
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.
