Alternative Hapten Design for Zearalenone Immunoreagent Generation.

Antonio Abad-Fuentes,Ismael Navarro-Fuertes,Antonio Abad-Somovilla,Josep Vicent Mercader,Consuelo Agulló,Daniel López-Puertollano

doi:10.3390/toxins14030185

Antonio Abad-Fuentes, Ismael Navarro-Fuertes + Show 4 more

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https://doi.org/10.3390/toxins14030185

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Abstract

Appropriate hapten design and synthesis have been identified as critical steps to generate high-performance immunoreagents and to develop sensitive and selective immunoanalytical methods. Antibodies and immunoassays for the major mycotoxin zearalenone have been reported and marketed. However, zearalenone haptens have mostly been prepared by the oxime active ester technique, and hapten characterization has generally been poor or non-existent. In the present study, novel haptens of zearalenone with longer linkers and with alternative tethering sites have been designed for immunizing and assay conjugate preparation. All of these molecules were purified and spectroscopically verified, and a structure-activity relationship evaluation was carried out. This approach revealed that the hapten with the linker at the carbonyl group generated antibodies with a higher affinity than the hapten functionalized at the phenyl moiety. Antibodies produced with the latter hapten, on the other hand, showed lower cross-reactivity values to the major zearalenone metabolites. Finally, similar immunoassay sensitivity was achieved with all of the antibodies when heterologous haptens were employed. Furthermore, by altering the structure of the competing antigen, the immunoassay selectivity was modified. These results demonstrate that immunochemical methods for zearalenone rapid analysis can still be improved in terms of sensitivity and selectivity.

Highlights

Mycotoxins are the group of chemical contaminants potentially present in food and feed for which immunochemical techniques enjoy a higher degree of implementation and acceptability in analytical laboratories around the world, even being supported and recommended by regulatory authorities in certain cases
Hapten ZEp generated more specific antibodies than hapten ZEo. These results demonstrate the major influence of the linker tethering site on the affinity and specificity of ZEN antibodies
Antibodies to zearalenone that were raised using hapten ZEo—with the spacer arm distal to the aromatic moiety of the mycotoxin—showed higher affinity than those obtained from hapten ZEp, displaying the aliphatic macrocyclic ring of the molecule

Summary

Introduction

Mycotoxins are the group of chemical contaminants potentially present in food and feed for which immunochemical techniques enjoy a higher degree of implementation and acceptability in analytical laboratories around the world, even being supported and recommended by regulatory authorities in certain cases. Immunoanalytical methods are based on the high-affinity, selective, reversible, and non-covalent binding between a target substance (analyte) and an antibody. In order to generate antibodies to low molecular weight compounds like mycotoxins, the target molecule must be covalently coupled to an immunogenic macromolecule, such as a protein. To prepare such a bioconjugate, the target compound must be chemically modified by introducing a functionalized aliphatic linker at specific positions as a protein. To prepare such a bioconjugate, the target compound must be chemically modified by introducing a functionalized aliphatic linker at specific positions of the molecular framework. The structural and electronic similarity between the synthetic functionalized analogue, so-called framework

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