Analytical and Bioanalytical Chemistry

, Volume 411, Issue 25, pp 6755–6765 | Cite as

Production of a specific monoclonal antibody and a sensitive immunoassay for the detection of diphacinone in biological samples

  • Hongfang Li
  • Shuang Liu
  • Baolei Dong
  • Chenglong Li
  • Huijuan Yang
  • Xiya Zhang
  • Kai Wen
  • Xuezhi Yu
  • Wenbo Yu
  • Jianzhong Shen
  • Jiancheng LiEmail author
  • Zhanhui Wang
Research Paper


Diphacinone (DPN) is an extensively used anticoagulant rodenticide that is also considered a hazardous chemical, which poses a threat to nontarget species. DPN poisoning cases in humans or other species frequently occur, while rapid and sensitive detection methods are rarely reported. Thus, it is meaningful to develop an immunoassay for DPN detection with high sensitivity and specificity. In this study, a hapten was synthesized and then conjugated with carrier proteins to prepare the immunogens with different conjugation ratios for the preparation of antibody. After evaluation of the antisera using an indirect competitive enzyme-linked immunosorbent assay (icELISA) and statistical analysis, we found that the immunogen prepared using the N,N-dicyclohexylcarbodiimide (DCC) method with a conjugation ratio of 28.5 could elicit mice to generate antibodies with high performance. Using hybridoma technology, we obtained the specific monoclonal antibody (mAb) 4G5 with a half maximal inhibitory concentration (IC50) of 0.82 ng/mL in buffer solution. We initially explored the recognition mechanism of DPN/CLDPN and mAb from both conformational and electronic aspects. Then, mAb 4G5 was applied to develop icELISA for biological samples. The limits of detection (LODs) of icELISA were 0.28 μg/L, 0.32 μg/L, and 0.55 μg/kg for swine plasma, urine, and liver samples, respectively, and the recoveries ranged from 72.3 to 103.3% with a coefficient of variation (CV) of less than 12.3% in spiked samples. In summary, we developed a sensitive, specific, and accurate icELISA for the detection of DPN in biological samples, which showed potential in food safety analysis and clinical diagnosis.

Graphical abstract


Diphacinone Conjugation methods High specificity Monoclonal antibody icELISA 


Funding information

This work is supported by the Beijing Municipal Science and Technology Commission (D171100008317003) and National Key R&D Program of China (2018YFC1602900).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The laboratory animals (mice) used in our experiments were in accordance with the relevant Chinese laws and according to the China Agriculture University regulations concerning protection of animals used for scientific purposes (2010-SYXK-0037). The mice used in this study were approved by the Ethics Committee on Experimental Animals and Animal Tests of China Agricultural University. The review number is AW23107102-2.

Supplementary material

216_2019_2051_MOESM1_ESM.pdf (400 kb)
ESM 1 (PDF 400 kb)


  1. 1.
    Tang J, Qi S, Chen X. Spectroscopic studies of the interaction of anti-coagulant rodenticide diphacinone with human serum albumin. J Mol Struct. 2005;779:87–95.CrossRefGoogle Scholar
  2. 2.
    Leporati M, Salomone A, Golè G, Vincenti M. Determination of anticoagulant rodenticides and α-chloralose in human hair. Application to a real case. J Anal Toxicol. 2016;40:277–85.CrossRefGoogle Scholar
  3. 3.
    Sudakin DL. Hamilton & Hardy’s industrial toxicology: rodenticides. 6th ed. New York: Wiley press; 2015.Google Scholar
  4. 4.
    Rattner BA, Horak KE, Warner SE, Johnston JJ. Acute toxicity of diphacinone in northern bobwhite: effects on survival and blood clotting. Ecotoxicol Environ Saf. 2010;73:1159–64.CrossRefGoogle Scholar
  5. 5.
    Yu CC, Atallah YH, Whitacre DM. Metabolism and disposition of diphacinone in rats and mice. Drug Metab Dispos. 1982;10:645–8.Google Scholar
  6. 6.
    King N, Tran MH. Long-acting anticoagulant rodenticide (superwarfarin) poisoning: a review of its historical development, epidemiology, and clinical management. Transfus Med Rev. 2015;29:250–8.CrossRefGoogle Scholar
  7. 7.
    Valchev I, Binev R, Yordanova V, Nikolov Y. Anticoagulant rodenticide intoxication in animals–a review. Turk J Vet Anim Sci. 2008;32:237–43.Google Scholar
  8. 8.
    Pitt WC, Higashi M, Primus TM. The effect of cooking on diphacinone residues related to human consumption of feral pig tissues. Food Chem Toxicol. 2011;49:2030–4.CrossRefGoogle Scholar
  9. 9.
    Lefebvre S, Fourel I, Queffélec S, Vodovar D, Megarbane B, Benoit E, et al. Poisoning by anticoagulant rodenticides in humans and animals: causes and consequences. 2017.
  10. 10.
    Lohr MT, Davis RA. Anticoagulant rodenticide use, non-target impacts and regulation: a case study from Australia. Sci Total Environ. 2018;634:1372–84.CrossRefGoogle Scholar
  11. 11.
    Gulati S, Gulati A. Anticoagulant rodenticide poisoning. Indian J Med Spec. 2018.
  12. 12.
    Chen J, Li YQ, Mai JP, Cao M. Rapid detection of serum diphenadione with gas chromatography–mass spectrometry. Chin Occup Med. 2007;34:491–2.Google Scholar
  13. 13.
    Jin M, Chen X, Ye M, Zhu Y. Analysis of indandione anticoagulant rodenticides in animal liver by eluent generator reagent free ion chromatography coupled with electrospray mass spectrometry. J Chromatogr A. 2008;1213:77–82.CrossRefGoogle Scholar
  14. 14.
    Smith LL, Liang B, Booth MC, Filigenzi MS, Tkachenko A, Gaskill CL. Development and validation of quantitative ultraperformance liquid chromatography–tandem mass spectrometry assay for anticoagulant rodenticides in liver. J Agric Food Chem. 2017;65:6682–91.CrossRefGoogle Scholar
  15. 15.
    Dong B, Zhao S, Li H, Wen K, Ke Y, Shen J, et al. Design, synthesis and characterization of tracers and development of a fluorescence polarization immunoassay for the rapid detection of ractopamine in pork. Food Chem. 2019;271:9–17.CrossRefGoogle Scholar
  16. 16.
    Mount ME, Kurth HJ, Jackson DY. Production of antibodies and development of an immunoassay for the anticoagulant, diphacinone. J Immunoass. 1988;9:69–81.CrossRefGoogle Scholar
  17. 17.
    Kurth MJ, Bruins P, Mount ME. Diphacinone: 13C NMR as a predictive tool in tricarbonyl chemistry. Tetrahedron Lett. 1985;26:4883–6.CrossRefGoogle Scholar
  18. 18.
    Yang H, Dai R, Zhang H, Zhang X, Shen J, Wen K, et al. Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water. Anal Bioanal Chem. 2016;408:6037–44.CrossRefGoogle Scholar
  19. 19.
    Bui QA, Vu THH, Ngo VKT, Kennedy IR, Lee NA, Allan R. Development of an ELISA to detect clenbuterol in swine products using a new approach for hapten design. Anal Bioanal Chem. 2016;408:6045–52.CrossRefGoogle Scholar
  20. 20.
    Wang F, Wang H, Shen Y, Yong L, Dong J, Xu Z, et al. Bispecific monoclonal antibody-based multianalyte ELISA for furaltadone metabolite, malachite green, and leucomalachite green in aquatic products. J Agric Food Chem. 2016;64:8054–61.CrossRefGoogle Scholar
  21. 21.
    Zhang X, Wen K, Wang Z, Jiang H, Beier RC, Shen J. An ultra−sensitive monoclonal antibody−based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk. Food Control. 2016;60:588–95.CrossRefGoogle Scholar
  22. 22.
    Ceballos-Alcantarilla E, Agulló C, Abad-Somovilla A, Abad-Fuentesb A, Mercader JV. Highly sensitive monoclonal antibody-based immunoassays for the analysis of fluopyram in food samples. Food Chem. 2019;288:117–26.CrossRefGoogle Scholar
  23. 23.
    Mercader JV, Abad-Somovilla A, Agulló C, Abad-Fuentes A. Fluxapyroxad haptens and antibodies for highly sensitive immunoanalysis of food samples. J Agric Food Chem. 2017;65:9333–41.CrossRefGoogle Scholar
  24. 24.
    Li H, Ma S, Zhang X, Zhang X, Li C, Dong B, et al. Generic hapten synthesis, broad-specificity monoclonal antibodies preparation, and ultrasensitive ELISA for five antibacterial synergists in chicken and milk. J Agric Food Chem. 2018;66:11170–9.CrossRefGoogle Scholar
  25. 25.
    Li H, Zhou P, Zhang J, Li D, Li X, Gao X. A theoretical guide for screening ionic liquid extractants applied in the separation of a binary alcohol-ester azeotrope through a DFT method. J Mol Liq. 2018;251:51–60.CrossRefGoogle Scholar
  26. 26.
    Zhang F, Liu B, Liu G, Zhang Y, Wang J, Wang S. Substructure−activity relationship studies on antibody recognition for phenylurea compounds using competitive immunoassay and computational chemistry. Sci Rep. 2018;8:3131.CrossRefGoogle Scholar
  27. 27.
    Jayasheela K, Al-Wahaibi LH, Periandy S, Hassan HM, Sebastian S. Probing vibrational activities, electronic properties, molecular docking and Hirshfeld surfaces analysis of 4-chlorophenyl ({[(1E)-3-(1H-imidazol-1-yl)-1-phenylpropylidene] amino} oxy) methanone: a promising anti-Candida agent. J Mol Struct. 2018;1159:83–95.CrossRefGoogle Scholar
  28. 28.
    Liu Z, Rigger L, Rossi JC, Sutherland JD, Pascal R. Mixed anhydride intermediates in the reaction of 5 (4H)-oxazolones with phosphate esters and nucleotides. Chem Eur J. 2016;22:14940–9.CrossRefGoogle Scholar
  29. 29.
    Hermanson GT. Bioconjugate techniques. 3rd ed. London: Academic press; 2013.Google Scholar
  30. 30.
    Hye YY, Park SW, Chung IM, Yi-Sook Jung YS. Anti-platelet effects of yuzu extract and its component. Food Chem Toxicol. 2011;49:3018–24.CrossRefGoogle Scholar
  31. 31.
    Jaschke AC, Honing H, Scherder EJA. Longitudinal analysis of music education on executive functions in primary school children. Front Neurosci. 2018;12:103.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Hongfang Li
    • 1
  • Shuang Liu
    • 1
  • Baolei Dong
    • 1
  • Chenglong Li
    • 1
  • Huijuan Yang
    • 1
  • Xiya Zhang
    • 2
  • Kai Wen
    • 1
  • Xuezhi Yu
    • 1
  • Wenbo Yu
    • 1
  • Jianzhong Shen
    • 1
  • Jiancheng Li
    • 1
    Email author
  • Zhanhui Wang
    • 1
  1. 1.Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Key Laboratory of Detection Technology for Animal–Derived Food Safety, Beijing Laboratory of Food Quality and SafetyChina Agricultural UniversityBeijingChina
  2. 2.College of Food Science and TechnologyHenan Agricultural UniversityZhengzhouChina

Personalised recommendations