High-affinity Antibodies from a Full Penthiopyrad-mimicking Hapten and Heterologous Immunoassay Development for Fruit Juice Analysis
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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.
KeywordsTarget mimicking Antibodies Fungicide residues ELISA Immunoassay
adult bovine serum
bovine serum albumin
enzyme-linked immunosorbent assay
goat antirabbit immunoglobulins conjugated to peroxidase
high-resolution mass spectrometry
limit of detection
maximum residue limit
PBS containing Tween 20
succinate dehydrogenase inhibitor
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.
Compliance with Ethical Standards
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.
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