Skip to main content
Log in

High-affinity Antibodies from a Full Penthiopyrad-mimicking Hapten and Heterologous Immunoassay Development for Fruit Juice Analysis

  • Published:
Food Analytical Methods Aims and scope Submit manuscript


New-generation succinate dehydrogenase inhibitors with increased antifungal performance have been developed during the last years. Particularly, penthiopyrad has been recently introduced in the agrochemical market as a complementary fungicide for combined treatments with other active principles displaying a different mode of action. In the present study, rapid and high-throughput analytical methods for penthiopyrad residue determination in foodstuffs were developed. A novel functionalized hapten that completely mimics the molecule of penthiopyrad was designed for high-affinity and specific antibody generation. Moreover, heterologous haptens were synthesized, and the influence of their respective molecular modifications over antibody binding was evaluated by competitive enzyme-linked immunosorbent assay. Two immunoassays were optimized using alternative assay formats, affording limits of detection for penthiopyrad in buffer around 0.07 ng/mL in both cases. Performance, in terms of accuracy and precision, of the developed immunochemical methods was studied using apple juice and red and white grape juices. The obtained results showed that penthiopyrad could be determined in those food samples in compliance with European maximum residue limits.

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

Access this article

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

Instant access to the full article PDF.

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

Similar content being viewed by others



adult bovine serum


bovine serum albumin




N,N′-disuccinimidyl carbonate


enzyme-linked immunosorbent assay


electrospray ionization


goat antirabbit immunoglobulins conjugated to peroxidase


high-resolution mass spectrometry


horseradish peroxidase




limit of detection


molar ratio


maximum residue limit




phosphate buffer


phosphate-buffered saline


PBS containing Tween 20


succinate dehydrogenase inhibitor


thin-layer chromatography




  • Abad-Fuentes A, Ceballos-Alcantarilla E, Mercader JV, Agulló C, Abad-Somovilla A, Esteve-Turrillas FA (2015) Determination of succinate-dehydrogenase-inhibitor fungicide residues in fruits and vegetables by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 407:4207–4211

    Article  CAS  Google Scholar 

  • AgroNews. (2017) Global SDHI fungicide market expected to reach $4 bn in 2020. Accessed 22 May 2017

  • Ahn KC, Kim HJ, McCoy MR, Gee SJ, Hammock BD (2011) Immunoassays and biosensors for monitoring environmental and human exposure to pyrethroid insecticides. J Agric Food Chem 59:2792–2802

    Article  CAS  Google Scholar 

  • APVMA, Australian Pesticides and Veterinary Medicines Authority (2012) Public release summary on the evaluation of penthiopyrad in the new product DuPont™ Fontelis® fungicide. APVMA Product Number 65100

  • Armarego WLF, Perrin DD (1997) In: purification of laboratory chemicals, 4th edn. Oxford, Butterworth-Heinemann Ltd

    Google Scholar 

  • Avenot HF, Michailides TJ (2010) Progress in understanding molecular mechanisms and evolution of resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in phytopathogenic fungi. Crop Prot 29:643–651

    Article  CAS  Google Scholar 

  • Ceballos-Alcantarilla E, Abad-Fuentes A, Aloisio V, Agulló C, Abad-Somovilla A, Mercader JV (2014) Haptens, bioconjugates, and antibodies for penthiopyrad immunosensing. Analyst 139:5338–5361

    Article  Google Scholar 

  • Culbreath AK, Brenneman TB, Kemerait RC Jr, Hammes GG (2009) Effect of the new pyrazole carboxamide fungicide penthiopyrad on late leaf spot and stem rot of peanut. Pest Manag Sci 65:66–73

    Article  CAS  Google Scholar 

  • Dankwardt A (2006) Immunochemical assays in pesticide analysis. In: Meyers RA (ed) Encyclopedia of analytical chemistry. John Wiley & Sons Ltd, Chichester, UK, pp 1–27

    Google Scholar 

  • DG SANCO (2013) Final review report for the active substance penthiopyrad. Document number SANCO/12078/2013 rev 2

  • EFSA, European Food Safety Authority (2013) Conclusion on the peer review of the pesticide risk assessment of the active substance penthiopyrad. EFSA J 11(2):3111

    Article  Google Scholar 

  • Esteve-Turrillas FA, Parra J, Abad-Fuentes A, Agulló C, Abad-Somovilla A, Mercader JV (2010) Hapten synthesis, monoclonal antibody generation, and development of competitive immunoassays for the analysis of picoxystrobin in beer. Anal Chim Acta 682:93–103

    Article  CAS  Google Scholar 

  • EU Pesticides Database. (2017) Accessed 22 May 2017

  • FAO (2011) Penthiopyrad. In: Joint FAO/WHO Meeting on Pesticide Residues. Pesticide Residues in Food 2011. FAO Plant Production and Protection Paper 211:189–194

    Google Scholar 

  • Gudmestad NC, Arabiat S, Miller JS, Pasche JS (2013) Prevalence and impact of SDHI fungicide resistance in Alternaria solani. Plant Dis 97(7):952–960

    Article  CAS  Google Scholar 

  • Gulkowska A, Buerge IJ, Poiger T (2014) Online solid phase extraction LC-MS/MS method for the analysis of succinate dehydrogenase inhibitor fungicides and its applicability to surface water samples. Anal Bioanal Chem 406:6419–6427

    Article  CAS  Google Scholar 

  • Li YF, Sun YM, Beier RC, Lei HT, Gee S, Hammock BD, Wang H, Wang Z, Sun X, Shen YD, Yang JY, Xu ZL (2017) Immunochemical techniques for multianalyte analysis of chemical residues in food and the environment: a review. TRAC-Trend Anal Chem 88:25–40

    Article  CAS  Google Scholar 

  • López-Moreno R, Mercader JV, Agulló C, Abad-Somovilla A, Abad-Fuentes A (2013) Structure–immunogenicity relationship of kresoxim-methyl regioisomeric haptens. Org Biomol Chem 11:7361–7371

    Article  Google Scholar 

  • Pesticide Properties Database. (2017) Accessed 22 May 2017

  • Proffer TJ, Lizotte E, Rothwell NL, Sundin GW (2013) Evaluation of dodine, fluopyram and penthiopyrad for the management of leaf spot and powdery mildew of tart cherry, and fungicide sensitivity screening of Michigan populations of Blumeriella jaapii. Pest Manag Sci 69:747–754

    Article  CAS  Google Scholar 

  • Sakurai S, Hagiwara H, Yanase Y (2011) Biological activity of penthiopyrad and study on sensitivity test for several plant pathogens. J Pestic Sci 36(4):520–523

    Article  CAS  Google Scholar 

  • Suri CR, Boro R, Nangia Y, Gandhi S, Sharma P, Wangoo N, Rajesh K (2009) Immunoanalytical techniques for analyzing pesticides in the environment. TRAC-Trend Anal Chem 28:29–39

    Article  CAS  Google Scholar 

  • Van Emon JM, Chuang JC, Dill K, Xiong G (2008) Immunoassays and biosensors. In: Tadeo JL (ed) Analysis of pesticides in food and environmental samples. CRC Press, Taylor & Francis Group, Boca Raton, pp 95–123

    Google Scholar 

  • Xu ZL, Shen YD, Beier RC, Yang JY, Lei HT, Wang H, Sun YM (2009) Application of computer-assisted molecular modeling for immunoassay of low molecular weight food contaminants: a review. Anal Chim Acta 647:125–136

    Article  CAS  Google Scholar 

  • Yoshikawa Y, Katsuta H, Kishi J, Yanase Y (2011) Structure–activity relationship of carboxin-related carboxamides as fungicide. J Pestic Sci 36(3):347–356

    Article  CAS  Google Scholar 

Download references


This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2012-39965-C02 and AGL2015-64488-C2) and cofinanced by FEDER funds. E.C.-A. is recipient of a predoctoral fellowship from the “Atracció de Talent, VLC-CAMPUS” program of the University of Valencia. Conjugate analysis was carried out at the Proteomics Unit—a member of ISCIII ProteoRedProteomics Platform—and animal manipulation was carried out at the Animal Production Section, both belonging to the SCSIE of the University of Valencia.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Josep V. Mercader.

Ethics declarations

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Conflict of Interest

Eric Ceballos-Alcantarilla declares that he has no conflict of interest. Consuelo Agulló declares that she has no conflict of interest. Antonio Abad-Fuentes declares that he has no conflict of interest. Mónica Escamilla-Aguilar declares that she has no conflict of interest. Antonio Abad-Somovilla declares that he has no conflict of interest. Josep V. Mercader declares that he has no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ceballos-Alcantarilla, E., Agulló, C., Abad-Fuentes, A. et al. High-affinity Antibodies from a Full Penthiopyrad-mimicking Hapten and Heterologous Immunoassay Development for Fruit Juice Analysis. Food Anal. Methods 10, 4013–4023 (2017).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: