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Development of a fast ELISA for the specific detection of both leucomalachite green and malachite green

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Abstract

Malachite green (MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms (LMG) that may reside in fish muscles for a long period, thus being harmful to human health. The aim of this study was to develop a competitive and direct enzyme-linked immunosorbent assay (ELISA) to detect MG and LMG specifically. The monoclonal antibody (mAb) to LMG was generated using a hybridoma technique. The obtained mAb showed good cross-reactivity (CR) to malachite green (MG), but not to crystal violet (CV) and Brilliant Green (BG). The mAb was used to develop a fast detecting ELISA of MG and LMG in fish. By introducing the conjugation LMG-HRP, the detection capability was 0.37 ng mL−1 for MG and LMG. The mean recovery from spiked grass carp tissues ranged from 76.2% to 82.9% and the coefficients of variation varied between 1.8% and 7.5%. The stable and efficient monoclonal cell line obtained is a sustainable source of sensitive and specific antibody to MG and LMG.

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References

  • Dowling, G., Mulder, P. P. J., Duffy, C., Regan, L., and Smyth, M. R., 2007. Confirmatory analysis of malachite green, leucomalachite green, crystal violet and leucocrystal violet in salmon by liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta, 586(1–2): 411–419.

    Article  Google Scholar 

  • Gong, P. F., Zhou, M. H., and Zheng, Z. Q., 2008. Used for testing the whole antigens of malachite green synthesis and identification. Veterinary Pharmaceuticals & Feed Additives, 23(6): 1–4.

    Google Scholar 

  • Hajee, C. A. J., and Haagsma, N., 1995. Simultaneous determination of malachite green and its metabolite leucomalachite green in eel plasma using post-column oxidation. Journal of Chromatography B, 669(2): 219–227.

    Article  Google Scholar 

  • Han, D., Yu, M., Knopp, D., Niessner, R., Wu, M., and Deng, A., 2007. Development of a highly sensitive and specific enzyme-linked immunosorbent assay for detection of Sudan I in food samples. Journal of Agricultural and Food Chemistry, 55(16): 6424–6430.

    Article  Google Scholar 

  • Holtzapple, C. K., Buckley, S. A., and Stanker, L. H., 1997. Production and characterization of monoclonal antibodies against sarafloxacin and cross-reactivity studies of related fluoroquinolones. Journal of Agricultural and Food Chemistry, 45: 1984–1990.

    Article  Google Scholar 

  • Hu, K., Huang, X. Y., Jiang, Y. S., Fang, W., and Yang, X. L., 2010. Monoclonal antibody based enzyme-linked immunosorbent assay for the specific detection of ciprofloxacin and enrofloxacin residues in fishery products. Aquaculture, 310: 8–12.

    Article  Google Scholar 

  • Huang, X. Y., Hu, K., Fang, W., Jin, Y., and Yang, X. L., 2008. Preparation of ciprofloxacin-carrier protein conjugation and identification of its products. Journal of Shanghai Fishery University, 17(5): 585–590.

    Google Scholar 

  • Long, C. Y., Mai, Z. B., Yang, Y. F., Zhu, B. H., Xu, X. M., Lu, L., and Zou, X. Y., 2009. Determination of multi-residue for malachite green, gentian violet and their metabolites in aquatic products by high-performance liquid chromatography coupled with molecularly imprinted solid-phase extraction. Journal of Chromatography A, 1216(12): 2275–2281.

    Article  Google Scholar 

  • Luo, X. B., Jiang, X., Tu, X. M., Luo, S. L., Yan, L. S., and Chen, B., 2010. Determination of malachite green in fish water samples by cloud-point extraction coupled to cation-selective exhaustive injection and sweeping-MEKC. Electrophoresis, 31(4): 688–694.

    Article  Google Scholar 

  • Mitrowska, K., Posyniak, A., and Zmudzki, J., 2007. The effects of cooking on residues of malachite green and leucomalachite green in carp muscles. Journal of Analytical chemistry, 586(1–2): 420–425.

    Google Scholar 

  • Mitrowska, K., Posyniak, A., and Zmudzki, J., 2005. Determination of malachite green and leucomalachite green in carp muscle by liquid chromatography with visible and fluorescence detection. Journal of Chromatography A, 1089(1–2): 187–192.

    Article  Google Scholar 

  • Oplatowskaa, M., Connollya, L., Stevensonb, P., Steadc, S., and Elliott, C. T., 2011. Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish. Analytica Chimica Acta, 698: 51–60.

    Article  Google Scholar 

  • Shen, Y. D., Deng, X. F., Xu, Z. L., Wang, Y., Lei, H. T., Wang, H., Yang, J. Y., Xiao, Z. L., and Sun, Y. M., 2011. Simultaneous determination of malachite green, brilliant green and crystal violet in grass carp tissues by a broad-specificity indirect competitive enzyme-linked immunosorbent assay. Analytica Chimica Acta, 707: 48–152.

    Article  Google Scholar 

  • Shen, Y. D., Wang, Y., Sun, Y. M., Lei, H. T., Wang, H., and Xiao, Z. L., 2008. Design, synthesis and identification of leucomalachite green haptens and complete antigens. Food Science, 29(7): 263–266.

    Google Scholar 

  • Stead, S. L., Ashwin, H., Johnston, B., Dallas, A., Kazakov, S. A., Tarbin, J. A., Sharman, M., Kay, J., and Keely, B. J., 2010. An RNA-aptamer-based assay for the detection and analysis of malachite green and leucomalachite green residues in fish tissue. Analytical Chemistry, 82(7): 2652–2660.

    Article  Google Scholar 

  • Tarbin, J. A., Barnes, K. A., Bygrave, J., and Farrington, W. H., 1998. Screening and confirmation of triphenylmethane dyes and their leuco metabolites in trout muscle using HPLC-Vis and ESP-LC-MS. Analyst, 123(12): 2567–2571.

    Article  Google Scholar 

  • Tao, Y. F., Chen, D. M., Chao, X. Q., Yu, H., Pan, Y. H., Liu, Z. L., Huang, L. L., Wang, Y. L., and Yuan, Z. H., 2011. Simultaneous determination of malachite green, gentian violet and their leuco-metabolites in shrimp and salmon by liquid chromatography-tandem mass spectrometry with accelerated solvent extraction and auto solid-phase clean-up. Food Control, 22(8): 246–1252.

    Google Scholar 

  • Tsai, C. H., Lin, J. D., and Lin, C. H., 2007. Optimization of the separation of malachite green in water by capillary electrophoresis Raman spectroscopy (CE-RS) based on the stacking and sweeping modes. Talanta, 72(2): 368–372.

    Article  Google Scholar 

  • Wang, Z. H., Zhu, Y., Ding, S. Y., He, F. Y., Beier, R. C., Li, J. C., Jiang, H. Y., Feng, C. W., Wan, Y. P., Zhang, S. X., Kai, Z. P., Yang, X. L., and Shen, J. Z., 2007. Development of a monoclonal antibody-based broad-specificity ELISA for fluoroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds. Analytical Chemistry, 79: 4471–4483.

    Article  Google Scholar 

  • Wang, M., Wang, Q., Cai, X. L., Wang, X. C., and Wu, J. J., 2009. Preparation and identification of monoclonal antibody of leuco malachite green. Animal Husbandry and Feed Science, 30(3): 1–3.

    Google Scholar 

  • Xing, W., He, L., Yang, H., Sun, C., Li, D., Yang, X., Li, Y., and Deng, A., 2009. Development of a sensitive and group-specific polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of malachite green and leucomalachite green in water and fish samples. Journal of the Science of Food and Agriculture, 89(13): 2165–2173.

    Article  Google Scholar 

  • Xing, W. W., Wang, R. M., Wang, J. Q., Yang, H., He, L., and Den, A. P., 2010. Sensitive enzyme-linked immunosorbent assay for determination of malachite green and leucomalachite green in fish and fishpond water samples. Chemical Research and Application, 1: 2–46.

    Google Scholar 

  • Yang, M. C., Fang, J. M., Kuo, T. F., Wang, D. M., Huang, Y. L., Liu, L. Y., Chen, P. H., and Chang, T. H., 2007. Production of antibodies for selective detection of malachite green and the related triphenylmethane dyes in fish and fishpond water. Journal of Agricultural and Food Chemistry, 55(22): 8851–8856.

    Article  Google Scholar 

  • Yang, J. L., Chen, P. J., Li, Z. G., Zhao, D. H., and Zhou, Q., 2010. Optimization of HPLC determination of malachite green residue in aquatic products. South China Fisheries Science, 6(4): 44–48.

    Google Scholar 

  • Zhao, C. C., Liu, Y. J., Xu, B. X., Yang, T. T., Wu, J., Shen, W. J., Zhang, L. S., Sun, X. L., and Zhao, X. L., 2009. Preparation and identification of anti-leucomalachite green antibodies. Chinese Journal of Health Laboratory Technology, 19(1): 39–41.

    Google Scholar 

  • Zhou, Y., Li, Y. S., Pan, F. G., Zhang, Y. Y., Lu, S. Y., Ren, H. L., Li, Z. H., Liu, Z. H., and Zhang, J. H., 2009. Development of a new monoclonal antibody based direct competitive enzyme-linked immunosorbent assay for detection of brevetoxins in food samples. Food Chemistry, 118(2): 467–471.

    Article  Google Scholar 

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Authors and Affiliations

  1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, 201306, P. R. China

    Yousheng Jiang, Li Chen, Kun Hu, Wenjuan Yu, Xianle Yang & Liqun Lu

Authors
  1. Yousheng Jiang
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  2. Li Chen
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  3. Kun Hu
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  4. Wenjuan Yu
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  5. Xianle Yang
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  6. Liqun Lu
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Correspondence to Yousheng Jiang.

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These authors contributed equally to this study.

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Jiang, Y., Chen, L., Hu, K. et al. Development of a fast ELISA for the specific detection of both leucomalachite green and malachite green. J. Ocean Univ. China 14, 340–344 (2015). https://doi.org/10.1007/s11802-015-2407-5

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  • DOI: https://doi.org/10.1007/s11802-015-2407-5

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