Development and validation of a direct competitive monoclonal antibody-based immunoassay for the sensitive and selective analysis of the phytoregulator forchlorfenuron

Abstract

Forchlorfenuron is a synthetic phytohormone with cytokinin-like activity used worldwide as a plant growth regulator to increase fruit size in a number of crops, mostly in kiwifruit and grape vines. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the determination of forchlorfenuron has been characterized and optimized. The selected immunoreagents afforded a highly selective assay with a limit of detection of 10 ng L−1 in buffer. This direct competitive ELISA was validated in terms of trueness, precision, and robustness using both commercial juice and whole fruit samples. Recoveries from fortified kiwifruit juices and white and red musts were between 97 % and 131 %, with relative standard deviations below 16 %. When homogenized whole fruits were analysed after acetonitrile extraction, recoveries between 96 % and 113 % were found, with a limit of quantification of 5 μg kg−1. The proposed immunoassay was validated by comparison with a reference chromatographic method using fruits from in-field treated grape and kiwifruit vines. Linear regression analysis of ELISA and HPLC–UV determinations showed an excellent correlation (r 2 = 0.998), whereas analysis of the slope (0.99 ± 0.01) and of the intercept (−1 ± 3) clearly proved that the developed competitive immunoassay provided results that were statistically comparable to those obtained by the instrumental method for the analysis of forchlorfenuron in fruits at trace levels.

Monoclonal ELISA validation for forchlorfenuron

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. 1.

    US Environmental Protection Agency; Office of prevention, pesticides, and toxic substances; Pesticide fact sheet: http://www.epa.gov/opprd001/factsheets/forchlorfenuron.pdf. Accessed February 15, 2012

  2. 2.

    Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, Miwa K, Yamashino T, Mizuno T (2001) Plant Cell Physiol 42:1017–1023

    Article  CAS  Google Scholar 

  3. 3.

    Kopečný D, Briozzo P, Popelková H, Marek S, Končitíková R, Spíchal L, Nisler J, Madzak C, Frébort I, Laloue M, Houba-Hérin N (2010) Biochimie 92:1052–1062

    Article  Google Scholar 

  4. 4.

    European Commission, Health & Consumer Protection Directorate General. SANCO/10392/2002 rev. 4 (2005)

  5. 5.

    EU pesticide database: http://ec.europa.eu/sanco_pesticides/. Accessed February 15, 2012

  6. 6.

    US Electronic Code of Federal Regulations; Title 40, “Protection of environment”; Part 180, “Tolerances and exemptions for pesticide chemical residues in food”; Subpart C, “Specific tolerances”; § 180.569, “Forchlorfenuron, tolerances for residues”. http://ecfr.gpoaccess.gov/. Accessed February 15, 2012

  7. 7.

    Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ (2003) J AOAC Int 86:412–431

    CAS  Google Scholar 

  8. 8.

    Mercader JV, Montoya A (1997) Anal Chim Acta 347:95–101

    Article  CAS  Google Scholar 

  9. 9.

    Salvador JP, Sánchez-Baeza F, Marco MP (2007) Anal Chem 79:3734–3740

    Article  CAS  Google Scholar 

  10. 10.

    Pastor-Navarro N, García-Bover C, Maquieira A, Puchades R (2004) Anal Bioanal Chem 379:1088–1099

    Article  CAS  Google Scholar 

  11. 11.

    Preston A, Fodey T, Elliott C (2008) Anal Chim Acta 608:178–185

    Article  CAS  Google Scholar 

  12. 12.

    Xu T, Wei KY, Wang J, Eremin SA, Liu SZ, Li QX, Li J (2010) Anal Biochem 405:41–49

    Article  CAS  Google Scholar 

  13. 13.

    Suárez-Pantaleón C, Mercader JV, Agulló C, Abad-Somovilla A, Abad-Fuentes A (2008) J Agric Food Chem 56:11122–11131

    Article  Google Scholar 

  14. 14.

    Suárez-Pantaleón C, Mercader JV, Agulló C, Abad-Somovilla A, Abad-Fuentes A (2010) J Agric Food Chem 58:8502–8511

    Article  Google Scholar 

  15. 15.

    Suárez-Pantaleón C, Mercader JV, Agulló C, Abad-Somovilla A, Abad-Fuentes A (2011) Org Biomol Chem 9:4863–4872

    Article  Google Scholar 

  16. 16.

    Esteve-Turrillas FA, Parra J, Abad-Fuentes A, Agulló C, Abad-Somovilla A, Mercader JV (2010) Anal Chim Acta 682:93–103

    Article  CAS  Google Scholar 

  17. 17.

    Hu JY, Li JZ (2006) J AOAC Int 89:1635–1640

    CAS  Google Scholar 

  18. 18.

    Moreira-Lima L, Barreiro EJ (2005) Curr Med Chem 12:23–49

    Google Scholar 

  19. 19.

    US Environmental Protection Agency; Office of Prevention, Pesticides, and Toxic Substances; R.E.D. Facts EPA-738-F-04-012: http://www.epa.gov/oppsrrd1/REDs/thidiazuron_factsheet.pdf. Accessed February 15, 2012

  20. 20.

    Esteve-Turrillas FA, Abad-Fuentes A, Mercader JV (2011) Food Chem 124:1727–1733

    Article  CAS  Google Scholar 

  21. 21.

    Parra J, Mercader JV, Agulló C, Abad-Somovilla A, Abad-Fuentes A (2012) Anal Chim Acta 715:105–112

    Article  CAS  Google Scholar 

  22. 22.

    Sharma D, Awasthi MD (2003) Chemosphere 50:589–594

    Article  CAS  Google Scholar 

  23. 23.

    Valverde A, Piedra L, Aguilera A, Boulaid M, Camacho F (2007) J Environ Sci Heal B 42:801–807

    Article  CAS  Google Scholar 

  24. 24.

    Lee SJ, Park HJ, Kim W, Jin JS, Abd El-Aty AM, Shim JH, Shin SC (2009) Biomed Chromatogr 23:434–442

    Article  CAS  Google Scholar 

  25. 25.

    Valverde A, Aguilera A, Ferrer C, Camacho F, Cammarano A (2010) J Agric Food Chem 58:2818–2823

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by Ministerio de Educación y Ciencia (PET2006_0423_00 and AGL2009-12940-C02/01/02/ALI), Ecología y Protección Agricola S.L., and FEDER Funds. C.S.-P., F.A.E.-T., and J.V.M. were hired by the CSIC under a predoctoral I3P contract, a postdoctoral JAE contract, and a Ramón y Cajal contract, respectively. All contracts were cofinanced by Ministerio de Ciencia e Innovación and the European Social Fund. We thank Laura López-Sánchez and Ana Izquierdo-Gil for the excellent technical assistance.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Antonio Abad-Fuentes.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 290 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Suárez-Pantaleón, C., Esteve-Turrillas, F.A., Mercader, J.V. et al. Development and validation of a direct competitive monoclonal antibody-based immunoassay for the sensitive and selective analysis of the phytoregulator forchlorfenuron. Anal Bioanal Chem 403, 2019–2026 (2012). https://doi.org/10.1007/s00216-012-5991-x

Download citation

Keywords

  • CPPU
  • Cytokinin
  • Hapten
  • ELISA
  • Food safety
  • HPLC