High-affinity Antibodies from a Full Penthiopyrad-mimicking Hapten and Heterologous Immunoassay Development for Fruit Ju
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High-affinity Antibodies from a Full Penthiopyrad-mimicking Hapten and Heterologous Immunoassay Development for Fruit Juice Analysis Eric Ceballos-Alcantarilla 1 & Consuelo Agulló 1 & Antonio Abad-Fuentes 2 & Mónica Escamilla-Aguilar 2 & Antonio Abad-Somovilla 1 & Josep V. Mercader 2
Received: 22 May 2017 / Accepted: 14 June 2017 # Springer Science+Business Media, LLC 2017
Abstract 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.
Abbreviations ABS adult bovine serum BSA bovine serum albumin DMF N,N′-dimethylformamide DSC N,N′-disuccinimidyl carbonate ELISA enzyme-linked immunosorbent assay ESI electrospray ionization GARgoat antirabbit immunoglobulins conjugated to HRP peroxidase HRMS high-resolution mass spectrometry HRP horseradish peroxidase IR infrared LOD limit of detection MR molar ratio MRL maximum residue limit OVA ovalbumin PB phosphate buffer PBS phosphate-buffered saline PBST PBS containing Tween 20 SDHI succinate dehydrogenase inhibitor TLC thin-layer chromatography UV ultraviolet
Keywords Target mimicking . Antibodies . Fungicide residues . ELISA . Immunoassay
Introduction
* Josep V. Mercader [email protected] 1
Department of Organic Chemistry, Universitat de València, Doctor Moliner 50, 46100 Burjassot, Valencia, Spain
2
Department of Technologies for Food Preservation and Safety, Institute of Agrochemistry and Food Technology (IATA), Spanish National Research Council (CSIC), Agustí Escardino 7, 46980 Paterna, Valencia, Spain
Succinate dehydrogenase inhibitors (SDHI) are a four-decadeold class of fungicides, but it was not until the last decade when the new-generation compounds of this family have gained prominence in the agrochemical sector for their higher efficiency and potency against a wider number of plant pathogenic fungi (Avenot and Michailides 2010; AgroNews 2017). These active principles
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