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A toxin-free enzyme-linked immunosorbent assay for the analysis of aflatoxins based on a VHH surrogate standard

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Abstract

A toxin-free enzyme-linked immunosorbent assay (ELISA) for aflatoxins was developed using an anti-idiotype nanobody VHH 2–5 as surrogate standard. Anti-idiotype nanobody VHH 2–5 was generated by immunizing an alpaca with anti-aflatoxin monoclonal antibody 1C11. This assay was used to detect aflatoxins in agro-products after a simple extraction with 75 % methanol/H2O. Aflatoxin concentration was calculated by a two-step approach: the concentration of VHH 2–5 was first obtained by a four-parameter logistic regression from the detected absorbance value at 450 nm, and then converted to aflatoxin concentration by a linear equation. The assay exhibits a limit of detection (LOD) of 0.015 ng mL−1, which is better than or comparable with conventional immunoassays. The performance of our VHH surrogate-based ELISA was further validated with a high-performance liquid chromatography (HPLC) method for total aflatoxins determination in 20 naturally contaminated peanut samples, displaying a good correlation (R 2 = 0.988). In conclusion, the proposed assay represents a first example applying an anti-idiotype VHH antibody as a standard surrogate in ELISA. With the advantages of high stability and ease of production, the VHH antibody-based standard surrogate can be extended in the future to immunoassays for other highly toxic compounds.

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Acknowledgement

This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201203094), the Project of National Science & Technology Pillar Plan (2012BAB19B09), the National Natural Science Foundation of China (31171702, 31501560), and Fundamental Research Funds for the Central Universities (Z109021566).

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Authors and Affiliations

  1. Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, China

    Yanru Wang, Peiwu Li, Qi Zhang, Xiaofeng Hu & Wen Zhang

  2. College of Food Science and Engineering, Northwest A&F University, 28 Xinong Road, Yangling, Xianyang, Shaanxi, 712100, China

    Yanru Wang

  3. Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, Hubei, 430062, China

    Peiwu Li, Xiaofeng Hu & Wen Zhang

  4. Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan, Hubei, 430062, China

    Peiwu Li & Qi Zhang

  5. Laboratory of Risk Assessment for Oilseeds Products, Wuhan, Ministry of Agriculture, Wuhan, Hubei, 430062, China

    Peiwu Li, Qi Zhang & Xiaofeng Hu

  6. Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan, Hubei, 430062, China

    Peiwu Li & Wen Zhang

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  1. Yanru Wang
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Correspondence to Peiwu Li or Qi Zhang.

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Published in the topical collection Immunoanalysis for Environmental Monitoring and Human Health with guest editors Shirley J. Gee, Ivan R. Kennedy, Alice Lee, Hideo Ohkawa, Tippawan Prapamontol, and Ting Xu.

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Wang, Y., Li, P., Zhang, Q. et al. A toxin-free enzyme-linked immunosorbent assay for the analysis of aflatoxins based on a VHH surrogate standard. Anal Bioanal Chem 408, 6019–6026 (2016). https://doi.org/10.1007/s00216-016-9370-x

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  • DOI: https://doi.org/10.1007/s00216-016-9370-x

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