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Early warning systems for monitoring toxic events may benefit from the availability of monoclonal antibodies enabling the sensitive and specific detection of anatoxin-a, a cyanotoxin involved in numerous cases of animal poisoning resulting from toxic algal blooms in freshwaters. Through the synthesis of three functionalized derivatives of anatoxin-a, we have succeeded in generating the first-ever reported immunoreagents (bioconjugates and antibodies) suitable for the development of immunoanalytical approaches aimed at rapid and onsite detection of this harmful cyanotoxin., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of agrochemicals for crop protection may result in the presence of toxic residues in soils and aquatic environments, besides in foodstuffs. Most often just the parent compound is included in the definition of pesticide residue, even though chemicals resulting from biotransformation and degradation routes might also be of toxicological relevance. Azoxystrobin is a broad-spectrum systemic fungicide widely used worldwide to combat pathogenic fungi affecting plants. We herein report the synthesis and detailed chemical characterization of several of the most relevant metabolites and degradates of azoxystrobin. These compounds were further employed as ligands for screening a collection of monoclonal antibodies to azoxystrobin, which had been previously generated from haptens functionalized at different positions of the target chemical. As a result, an antibody was identified capable of binding, with subnanomolar affinity, not only azoxystrobin but also its main transformation products, such as the so-called acid and enol derivatives, as well as the azoxystrobin (Z)-isomer. The selected binder was demonstrated as a useful immunoreagent for the development of immunochemical assays as novel analytical tools for the qualitative determination of azoxystrobin and its metabolites and degradates., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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 IC(50) 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)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Pyraclostrobin belongs to a new generation of fungicides widely used to preserve high valuable crops. In the present study, three monoclonal antibodies with different affinities to this modern strobilurin have been evaluated for their usefulness in the production of immunoaffinity columns suitable for the solid-phase extraction, concentration, and clean-up of residues from food commodities. Different immunosorbents were produced and characterized in terms of antibody immobilization efficiency, immunosorbent binding capacity, optimum elution conditions, and reusability. Covalent coupling of the antibodies to Sepharose-CNBr gel took place with high yield (over 90%), whereas the immunosorbent efficacy to retain the analyte (from 28 to 68%) was shown to depend on the amount and type of antibody immobilized on the support. As a matter of fact, columns prepared with the monoclonal antibody PYs5#14 were able to selectively bound up to 53 μg of pyraclostrobin per gram of beads. Acetonitrile solutions were preferred over methanolic ones for analyte elution, and some immunosorbents could be reused at least 4-6 times provided that the amount of pyraclostrobin and the volume of sample did not overload the column. Effectiveness of the selected immunoaffinity column was evidenced by the development of an extraction procedure for pyraclostrobin residues from fruit juices and further determination by high-performance liquid chromatography with UV detection. A concentration factor of 50 times was achieved with the developed immunoaffinity column, which eventually resulted in a limit of quantification of 0.01 mg L(-1). Finally, quantitative recoveries were obtained on apple juice and red grape must samples spiked with pyraclostrobin from 0.01 to 1 mg L(-1)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This paper describes the original synthesis of a functionalized derivative of the fungicide picoxystrobin and the generation of the first reported monoclonal antibodies against this strobilurin pesticide. The synthetic hapten was prepared by total synthesis from commercial chemicals and incorporating the spacer arm through a carbon-carbon single bond. Also, to obtain the immunogen, an uncommon hapten activation strategy based on N,N'-disuccinimidyl carbonate was employed, affording high activation yields and clean and reproducible coupling results. With these immunoreagents, two enzyme-linked immunosorbent assays (ELISAs) were developed: a competitive one-step assay using the antibody-coated direct ELISA format and a competitive two-step assay with the conjugate-coated indirect ELISA procedure. Both immunoassays were characterized in terms of sensitivity, selectivity, tolerance to solvents and matrix effects, achieving limits of detection below 0.2 μgL(-1). The optimized assays were used for the determination of picoxystrobin residues in beer, with recovery values ranging between 90 and 121% for the direct assay and from 79 to 122% for the indirect assay., (Copyright © 2010 Elsevier B.V. All rights reserved.)