Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays |
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Authors: | Javier Parra Josep V Mercader Consuelo Agulló Antonio Abad-Somovilla Antonio Abad-Fuentes |
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Affiliation: | 1. Department of Biotechnology, IATA–CSIC, Agustí Escardino 7, 46980 Paterna, València, Spain;2. Department of Organic Chemistry, Universitat de València, Doctor Moliner 50, 46100 Burjassot, València, Spain |
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Abstract: | Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and heterologous antigens, and the immunoassay consisting of monoclonal antibody AZo6#49 and the coating conjugate OVA–AZb6, which displayed an IC50 value of 0.102 μg L−1 and a LOD of 0.017 μg L−1, was eventually optimized. The response to different pH and ionic strength conditions of the specific assay was studied using a biparametric approach. In addition, the influence of Tween 20 and organic solvents over the assay parameters was also evaluated. After optimization, the developed immunochemical assay was applied to the analysis of azoxystrobin in spiked juices of relevant fruits and vegetables, showing excellent recoveries between 2 and 500 μg L−1. |
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Keywords: | AZ azoxystrobin BSA bovine serum albumin CR cross-reactivity DMEM Dulbecco's modified Eagle's medium ELISA enzyme-linked immunosorbent assay FBS fetal bovine serum HAT hipoxanthine&ndash aminopterine&ndash thimidine HFCS hybridoma fusion and cloning supplement HT hipoxanthine&ndash thimidine i-cELISA indirect competitive ELISA LOD limit of detection LOQ limit of quantification mAb monoclonal antibody MRL maximum residue limit OVA ovalbumin PBS phosphate-buffered saline PBST phosphate-buffered saline with Tween 20 RAM&ndash HRP polyclonal rabbit anti-mouse immunoglobulin peroxidase conjugate |
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