Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad
Nowadays, instrumental methodologies and rapid bioanalytical techniques complement each other for the analysis of toxic chemical compounds. Fluxapyroxad was commercialized a few years ago as a fungicide and today it is being used worldwide to control a variety of pests. In the present study, the development of monoclonal antibody-based immunochemical methods for the analysis of this chemical in food samples was evaluated for the first time. Novel haptens were synthesized and protein bioconjugates were prepared. High-affinity and specific monoclonal antibodies to fluxapyroxad were generated from two haptens with alternative linker tethering sites. Haptens with linker site heterology and a structurally heterologous hapten with a minor modification of the molecule conformation and volume but with a significant alteration of the electronic density of the pyrazole moiety were evaluated for immunoassay development. Direct and indirect competitive immunoassays were characterized and optimized, showing IC50 values for fluxapyroxad of 0.14 and 0.05 ng mL−1, respectively. The combination of two heterologies was particularly adequate in the indirect format. The two developed immunoassays showed excellent recoveries and coefficients of variation in fluxapyroxad-fortified plums and four varieties of grapes. Finally, a good correlation was found between the indirect immunoassay and UPLC-MS/MS when fruit samples with incurred residues of fluxapyroxad were analyzed. These monoclonal antibody-based immunochemical methods hold great promise for fluxapyroxad monitoring.
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Royal Society of Chemistry (UK)
2018-10-23
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dig-iata-es-10261-1794592019-10-23T04:30:36Z Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad Ceballos Alcantarilla, Eric López Puertollano, Daniel Agulló, Consuelo Abad Fuentes, Antonio Mercader Badia, Josep Vicent Universidad de Valencia Ministerio de Economía y Competitividad (España) European Commission Nowadays, instrumental methodologies and rapid bioanalytical techniques complement each other for the analysis of toxic chemical compounds. Fluxapyroxad was commercialized a few years ago as a fungicide and today it is being used worldwide to control a variety of pests. In the present study, the development of monoclonal antibody-based immunochemical methods for the analysis of this chemical in food samples was evaluated for the first time. Novel haptens were synthesized and protein bioconjugates were prepared. High-affinity and specific monoclonal antibodies to fluxapyroxad were generated from two haptens with alternative linker tethering sites. Haptens with linker site heterology and a structurally heterologous hapten with a minor modification of the molecule conformation and volume but with a significant alteration of the electronic density of the pyrazole moiety were evaluated for immunoassay development. Direct and indirect competitive immunoassays were characterized and optimized, showing IC50 values for fluxapyroxad of 0.14 and 0.05 ng mL−1, respectively. The combination of two heterologies was particularly adequate in the indirect format. The two developed immunoassays showed excellent recoveries and coefficients of variation in fluxapyroxad-fortified plums and four varieties of grapes. Finally, a good correlation was found between the indirect immunoassay and UPLC-MS/MS when fruit samples with incurred residues of fluxapyroxad were analyzed. These monoclonal antibody-based immunochemical methods hold great promise for fluxapyroxad monitoring. This work was supported by the Spanish Ministerio de Economía y Competitividad (AGL2012-39965-C02 and AGL2015-64488-C2) and cofinanced by the European Regional Development Funds. E. C.-A. was the recipient of a predoctoral fellowship from the “Atracció de Talent, VLC-CAMPUS” program of the University of Valencia. D. L.-P. was supported by a FPU predoctoral grant (FPU14/05266) from the Ministerio de Educación, Cultura y Deporte, Spain. Conjugate analysis was carried out at the Proteomics Section, which belongs to ProteoRed, PRB2-3, and was supported by grant PT17/0019, of the PE I+D+i 2013-2016, and funded by ISCIII and ERDF, and animal manipulation was performed at the Animal Production Section, both services belonging to the SCSIE of the University of Valencia. Peer reviewed 2019-04-05T12:10:51Z 2019-04-05T12:10:51Z 2018-10-23 artículo http://purl.org/coar/resource_type/c_6501 Analyst 143 (23): 5718-5727 (2018) 0003-2654 http://hdl.handle.net/10261/179459 10.1039/c8an01771a http://dx.doi.org/10.13039/501100003329 http://dx.doi.org/10.13039/501100000780 en #PLACEHOLDER_PARENT_METADATA_VALUE# info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/AGL2015-64488-C2 Postprint https://doi.org/10.1039/c8an01771a Sí open Royal Society of Chemistry (UK) |
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Nowadays, instrumental methodologies and rapid bioanalytical techniques complement each other for the analysis of toxic chemical compounds. Fluxapyroxad was commercialized a few years ago as a fungicide and today it is being used worldwide to control a variety of pests. In the present study, the development of monoclonal antibody-based immunochemical methods for the analysis of this chemical in food samples was evaluated for the first time. Novel haptens were synthesized and protein bioconjugates were prepared. High-affinity and specific monoclonal antibodies to fluxapyroxad were generated from two haptens with alternative linker tethering sites. Haptens with linker site heterology and a structurally heterologous hapten with a minor modification of the molecule conformation and volume but with a significant alteration of the electronic density of the pyrazole moiety were evaluated for immunoassay development. Direct and indirect competitive immunoassays were characterized and optimized, showing IC50 values for fluxapyroxad of 0.14 and 0.05 ng mL−1, respectively. The combination of two heterologies was particularly adequate in the indirect format. The two developed immunoassays showed excellent recoveries and coefficients of variation in fluxapyroxad-fortified plums and four varieties of grapes. Finally, a good correlation was found between the indirect immunoassay and UPLC-MS/MS when fruit samples with incurred residues of fluxapyroxad were analyzed. These monoclonal antibody-based immunochemical methods hold great promise for fluxapyroxad monitoring. |
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Universidad de Valencia |
author_facet |
Universidad de Valencia Ceballos Alcantarilla, Eric López Puertollano, Daniel Agulló, Consuelo Abad Fuentes, Antonio Mercader Badia, Josep Vicent |
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artículo |
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Ceballos Alcantarilla, Eric López Puertollano, Daniel Agulló, Consuelo Abad Fuentes, Antonio Mercader Badia, Josep Vicent |
spellingShingle |
Ceballos Alcantarilla, Eric López Puertollano, Daniel Agulló, Consuelo Abad Fuentes, Antonio Mercader Badia, Josep Vicent Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad |
author_sort |
Ceballos Alcantarilla, Eric |
title |
Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad |
title_short |
Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad |
title_full |
Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad |
title_fullStr |
Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad |
title_full_unstemmed |
Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad |
title_sort |
combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad |
publisher |
Royal Society of Chemistry (UK) |
publishDate |
2018-10-23 |
url |
http://hdl.handle.net/10261/179459 http://dx.doi.org/10.13039/501100003329 http://dx.doi.org/10.13039/501100000780 |
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