Molecular Nutrition

Establishment of Immunologic Detection Method for Aflatoxin B1 Based on Preparation of Aflatoxin B1 Monoclonal Antibody

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  • 1. Key Laboratory of Animal Immunology of Ministry of Agriculture, Key Laboratory of Animal Immunology of Henan Academy of Agriculture Science, Zhengzhou 450002, China;
    2. Department of Urology, People's Hospital of Zhengzhou, Zhengzhou 450003, China;
    3. School of Life Science, Henan Agricultural University, Zhengzhou 450002, China;
    4. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China

Received date: 2018-08-02

  Online published: 2019-03-18

Abstract

In order to detect aflatoxin B1 (AFB1) content in cereals and feeds rapidly and correctly, a highly sensitive AFB1 monoclonal antibody was prepared and an indirect competitive enzyme-linked immunosorbent assay (ELISA) for AFB1 was established. AFB1 complete antigens[AFB1-bovine serum albumin (BSA) and AFB1-ovalbumin (OVA)] were prepared via carbodiimide (EDC) method and used to immunize the BALB/c mice. After the cell fusion, hybridoma cell line which could secret highly sensitive AFB1 monoclonal antibody was selected. Then a lot of AFB1 monoclonal antibodies were prepared using inducing ascites in vivo, and the AFB1 monoclonal antibody properties were identified. Finally, an indirect competitive ELISA method for detecting AFB1 based on AFB1 monoclonal antibody was established. A hybridoma cell line 9H1F5 was selected, and the titer of AFB1 monoclonal antibody was up to 5.12×105, the affinity constant (Ka)=6.72×107 L/mol, and the subtype was immunoglobulin G2 (IgG2) measured by indirect ELISA method. According to the indirect competitive ELISA, the median inhibitory concentration (IC50) of AFB1 was 0.232 ng/mL, the detection range was 0.014 to 1.920 ng/mL, and the limit of detection (LOD) of AFB1 was 0.014 ng/mL. Besides, cross reaction rates of AFB1 monoclonal antibody with aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1) were 40.21%, 33.19%, 31.96% and 4.40%, respectively, but there were no cross reactions with other mycotoxins. According to the above results, the highly sensitive and specific AFB1 monoclonal antibody is successfully prepared, and the indirect competitive ELISA method for AFB1 detection is established on the base of AFB1 monoclonal antibody, which can provide the foundation for AFB1 rapid immunological detection in cereals and feeds.

Cite this article

YAO Jingjing, HU Xiaofei, HAN Junling, XU Fan, TENG Man, XING Yunrui, SUN Yaning, DENG Ruiguang, ZHANG Gaiping . Establishment of Immunologic Detection Method for Aflatoxin B1 Based on Preparation of Aflatoxin B1 Monoclonal Antibody[J]. Chinese Journal of Animal Nutrition, 2019 , 31(3) : 1405 -1414 . DOI: 10.3969/j.issn.1006-267x.2019.03.048

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