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Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water

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Abstract

Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin–leucine–arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL−1) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 μg L−1, respectively, and a recovery of 62–86 % with a coefficient of variation below 12.6 % in water samples.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (U1301214).

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Authors and Affiliations

  1. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, China

    Huijuan Yang, Jianzhong Shen & Zhanhui Wang

  2. Beijing Laboratory for Food Quality and Safety, No. 2 Yuanmingyuan West Road, Beijing, 100193, China

    Rui Dai & Huiyan Zhang

  3. Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, No. 2 Yuanmingyuan West Road, Beijing, 100193, China

    Chenglong Li, Xiya Zhang & Kai Wen

Authors
  1. Huijuan Yang
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  2. Rui Dai
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  8. Zhanhui Wang
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Correspondence to Zhanhui Wang.

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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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Yang, H., Dai, R., Zhang, H. et al. Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water. Anal Bioanal Chem 408, 6037–6044 (2016). https://doi.org/10.1007/s00216-016-9692-8

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  • DOI: https://doi.org/10.1007/s00216-016-9692-8

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