Skip to main content
SpringerLink
Log in

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

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

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

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  3. Sudakin DL. Hamilton & Hardy’s industrial toxicology: rodenticides. 6th ed. New York: Wiley press; 2015.

    Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  5. Yu CC, Atallah YH, Whitacre DM. Metabolism and disposition of diphacinone in rats and mice. Drug Metab Dispos. 1982;10:645–8.

    CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.5772/intechopen.69955.

  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.

    Article  CAS  PubMed  Google Scholar 

  11. Gulati S, Gulati A. Anticoagulant rodenticide poisoning. Indian J Med Spec. 2018. https://doi.org/10.1016/j.injms.2018.04.010.

  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.

    CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  17. Kurth MJ, Bruins P, Mount ME. Diphacinone: 13C NMR as a predictive tool in tricarbonyl chemistry. Tetrahedron Lett. 1985;26:4883–6.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  29. Hermanson GT. Bioconjugate techniques. 3rd ed. London: Academic press; 2013.

    Google Scholar 

  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.

    Article  CAS  Google Scholar 

  31. Jaschke AC, Honing H, Scherder EJA. Longitudinal analysis of music education on executive functions in primary school children. Front Neurosci. 2018;12:103.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Funding

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

Author information

Authors and Affiliations

  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 Safety, China Agricultural University, Beijing, 100193, China

    Hongfang Li, Shuang Liu, Baolei Dong, Chenglong Li, Huijuan Yang, Kai Wen, Xuezhi Yu, Wenbo Yu, Jianzhong Shen, Jiancheng Li & Zhanhui Wang

  2. College of Food Science and Technology, Henan Agricultural University, Zhengzhou, 450002, Henan, China

    Xiya Zhang

Authors
  1. Hongfang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Baolei Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chenglong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Huijuan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiya Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kai Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xuezhi Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Wenbo Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jianzhong Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jiancheng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Zhanhui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jiancheng Li.

Ethics declarations

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.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 400 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, H., Liu, S., Dong, B. et al. Production of a specific monoclonal antibody and a sensitive immunoassay for the detection of diphacinone in biological samples. Anal Bioanal Chem 411, 6755–6765 (2019). https://doi.org/10.1007/s00216-019-02051-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-019-02051-2

Keywords

Search

Navigation