Analytical and Bioanalytical Chemistry

, Volume 410, Issue 28, pp 7263–7273 | Cite as

Binding properties of broad-specific monoclonal antibodies against three organophosphorus pesticides by a direct surface plasmon resonance immunosensor

  • Shasha Jiao
  • Pengyan Liu
  • Ying Liu
  • Rubing Zou
  • Ying Zhao
  • Yihua LiuEmail author
  • Guonian Zhu
  • Yirong GuoEmail author
Paper in Forefront


In this study, heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) was introduced into the screening of hybridomas for the development of broad-specific monoclonal antibodies (mAbs) against organophosphorus (OP) pesticides. After immunization, two formats of icELISA based on the homologous hapten antigen and four heterologous hapten antigens were conducted for hybridoma screening. Two mAbs 2G6 and 7B2 with good recognition toward three OP pesticides (parathion, methyl-parathion, and fenitrothion) were produced. Results of the icELISA showed that the two mAbs exhibited high sensitivity against three OP pesticides, with IC50 ranging from 2.93 to 19.71 ng mL−1. Moreover, a non-competitive surface plasmon resonance (SPR) immunosensor was used for characterizing the binding properties of the mAbs to OP pesticides. After kinetic analysis, equilibrium dissociation constant (KD) values of mAbs 2G6 and 7B2 were calculated as 1.45 × 10−9 M and 4.26 × 10−9 M for parathion, 6.75 × 10−9 M and 4.17 × 10−9 M for methyl-parathion, and 2.44 × 10−8 M and 1.19 × 10−8 M for fenitrothion, respectively. Whereas, both icELISA and SPR-based immunoassay indicated that the two mAbs could not recognize other five OP analogs. Since SPR-based immunoassay provides comprehensive information of two molecules directly interacting with each other, it is a valuable tool during the development and characterization of broad-specific mAbs.

Graphical abstract


OP pesticides Heterologous icELISA SPR immunosensor Broad-specific mAb 



This study was financially supported by National Key R&D Program of China (2017YFF0210200 and 2016YFD0201302) and the Agricultural Project for Public Technology Research in Zhejiang Province (2016C32004).

Compliance with ethical standards

All studies on mice were performed under the guidance of the animal welfare committee of Zhejiang University in China.

Conflict of interests

The authors have declared no conflict of interests.

Supplementary material

216_2018_1337_MOESM1_ESM.pdf (176 kb)
ESM 1 (PDF 175 kb)


  1. 1.
    Roex EWM, Keijzers R, van Gestel CAM. Acetylcholinesterase inhibition and increased food consumption rate in the zebrafish, Danio rerio, after chronic exposure to parathion. Aquat Toxicol. 2003;64:451–60.CrossRefPubMedGoogle Scholar
  2. 2.
    Zhao X, Kong W, Wei J, Yang M. Gas chromatography with flame photometric detection of 31 organophosphorus pesticide residues in Alpinia oxyphylla dried fruits. Food Chem. 2014;162:270–6.CrossRefPubMedGoogle Scholar
  3. 3.
    Harshit D, Charmy K, Nrupesh P. Organophosphorus pesticides determination by novel HPLC and spectrophotometric method. Food Chem. 2017;230:448–53.CrossRefPubMedGoogle Scholar
  4. 4.
    Andrade GCRM, Monteiro SH, Francisco JG, Figueiredo LA, Botelho RG, Tornisielo VL. Liquid chromatography–electrospray ionization tandem mass spectrometry and dynamic multiple reaction monitoring method for determining multiple pesticide residues in tomato. Food Chem. 2015;175:57–65.CrossRefPubMedGoogle Scholar
  5. 5.
    Liu Y, Liu R, Boroduleva A, Eremin S, Guo Y, Zhu G. A highly specific and sensitive fluorescence polarization immunoassay for the rapid detection of triazophos residue in agricultural products. Anal Methods. 2016;8:6636–44.CrossRefGoogle Scholar
  6. 6.
    Lan M, Guo Y, Zhao Y, Liu Y, Gui W, Zhu G. Multi-residue detection of pesticides using a sensitive immunochip assay based on nanogold enhancement. Anal Chim Acta. 2016;938:146–55.CrossRefPubMedGoogle Scholar
  7. 7.
    Zhao F, Hu C, Wang H, Zhao L, Yang Z. Development of a MAb-based immunoassay for the simultaneous determination of O,O-diethyl and O,O-dimethyl organophosphorus pesticides in vegetable and fruit samples pretreated with QuEChERS. Anal Bioanal Chem. 2015;407:8959–70.CrossRefPubMedGoogle Scholar
  8. 8.
    Xu Z, Sun W, Yang J, Jiang Y, Campbell K, Shen Y, et al. Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples. J Agric Food Chem. 2012;60:2069–75.CrossRefPubMedGoogle Scholar
  9. 9.
    Du P, Jin M, Yang L, Du X, Chen G, Zhang C, et al. A rapid immunomagnetic-bead-based immunoassay for triazophos analysis. RSC Adv. 2015;5:81046–51.CrossRefGoogle Scholar
  10. 10.
    Shi H, Li H, Hua X, Zheng Z, Zhu G, Wang M. Characterization of multihapten antigens on antibody sensitivity and specificity for parathion. Anal Lett. 2014;47:2699–707.CrossRefGoogle Scholar
  11. 11.
    Xu Z, Xie G, Li Y, Wang B, Beier RC, Lei H, et al. Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure–activity relationship study of antibody recognition. Anal Chim Acta. 2009;647:90–6.CrossRefPubMedGoogle Scholar
  12. 12.
    Liu B, Ge Y, Zhang Y, Song Y, Lv Y, Wang X, et al. Production of the class-specific antibody and development of direct competitive ELISA for multi-residue detection of organophosphorus pesticides. Food Agric Immunol. 2012;23:157–68.CrossRefGoogle Scholar
  13. 13.
    Piao YZ, Kim YJ, Kim YA, Lee H, Hammock BD, Lee YT. Development of ELISAs for the class-specific determination of organophosphorus pesticides. J Agric Food Chem. 2009;57:10004–13.CrossRefPubMedGoogle Scholar
  14. 14.
    Wang C, Li X, Liu Y, Guo Y, Xie R, Gui W, et al. Development of a Mab-based heterologous immunoassay for the broad-selective determination of organophosphorus pesticides. J Agric Food Chem. 2010;58:5658–63.CrossRefPubMedGoogle Scholar
  15. 15.
    Wang Z, Li N, Zhang S, Zhang H, Sheng Y, Shen J. Production of antibodies and development of enzyme-linked immunosorbent assay for valnemulin in porcine liver. Food Addit Contam: Part A. 2013;30:244–52.CrossRefGoogle Scholar
  16. 16.
    Guo Y, Sanders M, Galvita A, Heyerick A, Deforce D, Bracke M, et al. Heterologous screening of hybridomas for the development of broad-specific monoclonal antibodies against deoxynivalenol and its analogues. World Mycotoxin J. 2014;7:257–65.CrossRefGoogle Scholar
  17. 17.
    Chang AL, McKeague M, Liang JC, Smolke CD. Kinetic and equilibrium binding characterization of aptamers to small molecules using a label-free, sensitive, and scalable platform. Anal Chem. 2014;86:3273–8.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Scarano S, Mascini M, Turner APF, Minunni M. Surface plasmon resonance imaging for affinity-based biosensors. Biosens Bioelectron. 2010;25:957–66.CrossRefPubMedGoogle Scholar
  19. 19.
    Estevez MC, Belenguer J, Gomez-Montes S, Miralles J, Escuela AM, Montoya A, et al. Indirect competitive immunoassay for the detection of fungicide thiabendazole in whole orange samples by surface plasmon resonance. Analyst. 2012;137:5659–65.CrossRefPubMedGoogle Scholar
  20. 20.
    Mauriz E, García-Fernández C, Mercader JV, Abad-Fuentes A, Escuela AM, Lechuga LM. Direct surface plasmon resonance immunosensing of pyraclostrobin residues in untreated fruit juices. Anal Bioanal Chem. 2012;404:2877–86.PubMedGoogle Scholar
  21. 21.
    Hirakawa Y, Yamasaki T, Harada A, Ohtake T, Adachi K, Iwasa S, et al. Analysis of the fungicide boscalid in horticultural crops using an enzyme-linked immunosorbent assay and an immunosensor based on surface plasmon resonance. J Agric Food Chem. 2015;63:8075–82.CrossRefPubMedGoogle Scholar
  22. 22.
    Yakes BJ, Kanyuck KM, DeGrasse SL. First report of a direct surface plasmon resonance immunosensor for a small molecule seafood toxin. Anal Chem. 2014;86:9251–5.CrossRefPubMedGoogle Scholar
  23. 23.
    Guo Y, Liu R, Liu Y, Xiang D, Liu Y, Gui W, et al. A non-competitive surface plasmon resonance immunosensor for rapid detection of triazophos residue in environmental and agricultural samples. Sci Total Environ. 2018;613-614:783–91.CrossRefPubMedGoogle Scholar
  24. 24.
    Langer N, Steinicke F, Lindigkeit R, Ernst L, Beuerle T. Determination of cross-reactivity of poly- and monoclonal antibodies for synthetic cannabinoids by direct SPR and ELISA. Forensic Sci Int. 2017;280:25–34.CrossRefPubMedGoogle Scholar
  25. 25.
    Liu Y, Jin M, Gui W, Cheng J, Guo Y, Zhu G. Hapten design and indirect competitive immunoassay for parathion determination: correlation with molecular modeling and principal component analysis. Anal Chim Acta. 2007;591:173–82.CrossRefPubMedGoogle Scholar
  26. 26.
    McAdam DP, Hill AS, Beasley HL, Skerritt JH. Mono- and polyclonal antibodies to the organophosphate fenitrothion. 1. Approaches to hapten–protein conjugation. J Agric Food Chem. 1992;40:1466–70.CrossRefGoogle Scholar
  27. 27.
    Riggle B, Dunbar B. Development of enzyme immunoassay for the detection of the herbicide norflurazon. J Agric Food Chem. 1990;38:1922–5.CrossRefGoogle Scholar
  28. 28.
    Wang C, Liu Y, Guo Y, Liang C, Li X, Zhu G. Development of a McAb-based immunoassay for parathion and influence of the competitor structure. Food Chem. 2009;115:365–70.CrossRefGoogle Scholar
  29. 29.
    Cannon MJ, Papalia GA, Navratilova I, et al. Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology. Anal Biochem. 2004;330:98–113.CrossRefPubMedGoogle Scholar
  30. 30.
    Henniona M, Barcelo D. Strengths and limitations of immunoassays for effective and efficient use for pesticide analysis in water samples: a review. Anal Chim Acta. 1998;362:3–34.CrossRefGoogle Scholar
  31. 31.
    Kim YJ, Cho YA, Lee H, Lee YT. Investigation of the effect of hapten heterology on immunoassay sensitivity and development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Anal Chim Acta. 2003;494:29–40.CrossRefGoogle Scholar
  32. 32.
    Holthues H, Pfeifer-Fukumura U, Sound I, Baumann W. Evaluation of the concept of heterology in a monoclonal antibody-based ELISA utilizing direct hapten linkage to polystyrene microtiter plates. J Immunol Methods. 2005;304:68–77.CrossRefPubMedGoogle Scholar
  33. 33.
    Dong S, Zhang C, Zhang X, Liu Y, Zhong J, Xie Y, et al. Production and characterization of monoclonal antibody broadly recognizing Cry1 toxins by use of designed polypeptide as Hapten. Anal Chem. 2016;88:7023–32.CrossRefPubMedGoogle Scholar
  34. 34.
    Li X, Zhang H, Ji Y, Zheng Z, Bian Q, Zhu G. Immunochemical and molecular characteristics of monoclonal antibodies against organophosphorus pesticides and effect of hapten structures on immunoassay selectivity. Food Agric Immunol. 2013;26:109–19.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Shasha Jiao
    • 1
  • Pengyan Liu
    • 1
  • Ying Liu
    • 1
  • Rubing Zou
    • 1
  • Ying Zhao
    • 1
  • Yihua Liu
    • 1
    • 2
    Email author
  • Guonian Zhu
    • 1
  • Yirong Guo
    • 1
    Email author
  1. 1.Institute of Pesticide and Environmental Toxicology, Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and InsectsZhejiang UniversityZhejiangChina
  2. 2.Research Institute of Subtropical ForestryChinese Academy of ForestryZhejiangChina

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