Derivados funcionalizados e inmunorreactivos para el fungicida fludioxonil

La presente invención se refiere a haptenos, conjugados, derivados marcados y anticuerpos para fludioxonil. Asímismo, la presente invención también se refiere al uso de conjugados de fludioxonil como antígenos de ensayo o inmunógenos para obtener anticuerpos de este fungicida; y al uso de los derivados marcados de fludioxonil como antígenos de ensayo. Además, la presente invención también se refiere a un método de análisis de fludioxonil utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcados. Esta invención también proporciona un kit para analizar fludioxonil que comprende anticuerpos de este fungicida, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcadosPeer reviewedConsejo Superior de Investigaciones Científicas, Universitat de ValènciaB1 Patente sin examen previ

Derivados funcionalizados e inmunorreactivos para el fungicida fludioxonil

La presente invención se refiere a haptenos, conjugados, derivados marcados y anticuerpos para fludioxonil. Asímismo, la presente invención también se refiere al uso de conjugados de fludioxonil como antígenos de ensayo o inmunógenos para obtener anticuerpos de este fungicida; y al uso de los derivados marcados de fludioxonil como antígenos de ensayo. Además, la presente invención también se refiere a un método de análisis de fludioxonil utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcados. Esta invención también proporciona un kit para analizar fludioxonil que comprende anticuerpos de este fungicida, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcadosPeer reviewedConsejo Superior de Investigaciones Científicas, Universitat de ValènciaB1 Patente sin examen previ

Site-heterologous haptens and competitive monoclonal antibody-based immunoassays for pyrimethanil residue analysis in foodstuffs

Pyrimethanil is a widely used post-harvest fungicide whose residues are commonly found in fruits and vegetables. We herein report the synthesis of two new functionalized derivatives of pyrimethanil carrying an equivalent spacer arm at different positions of the molecule. The influence of the linker tethering site on the affinity of monoclonal antibodies was shown. Moreover, the development of competitive immunoassays in different formats and with high sensitivity to the target analyte - IC50 values below 0.3μg/L - is described. Optimized assays were characterized by the determination of the limit of quantification, trueness, and precision using water-diluted QuEChERS extracts of fruits and vegetables fortified with pyrimethanil. Finally, in-field treated and blind spiked strawberry, tomato, and cucumber samples were analyzed by the developed immunoassays and a reference chromatographic method. Using Deming regression analysis, we show that results from both methodologies were statistically comparable.This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2009-12940-C02-01-02/ALI) and cofinanced by FEDER funds. J.V.M. and F.A.E.-T. were hired by the Consejo Superior de Investigaciones Científicas (CSIC), the former under a Ramón y Cajal contract and the latter under a JAE-doc contract, both of them financed by Ministerio de Ciencia e Innovación and the European Social Fund.Peer Reviewe

Immunoassays for pyraclostrobin analysis in processed food products using novel monoclonal antibodies and QuEChERS-based extracts

Strobilurins are a family of synthetic natural-derived pesticides widely used for fungi control in many crops. As part of a larger research project, a collection of monoclonal antibodies to one of the most relevant strobilurins, i.e. pyraclostrobin, has been generated using functionalized haptens with the spacer arm located at different sites. The toxophore aliphatic moiety, as observed with other strobilurins, and the planar aromatic two-ring system characteristic of this molecule were revealed as the best tethering sites for high-affinity monoclonal antibody production. A central derivatization position did not generate a good immune response, but such derivatives were useful as heterologous competitors in order to improve assay sensitivity. Direct and indirect assays were characterized in terms of solvent influence, pH, and ionic strength. Immunoassays with limits of detection below 0.1 οg/L were developed. A QuEChERS-based procedure was applied to the extraction of blueberry, strawberry, raspberry, peach, apricot, and plum jams. Clean acetonitrile extracts were properly diluted in water and analyzed by both optimized immunoassays with good accuracy and precision at levels covering the European maximum residue limits for those fruits. © 2012 Elsevier Ltd.This work was supported by Ministerio de Educación y Ciencia (AGL2006-12750-C02-01/02/ALI) and by FEDER funds. J.V.M. was hired by the CSIC under a Ramón y Cajal contract financed by Ministerio de Ciencia e Innovación and the European Social Fund.Peer Reviewe

Derivados funcionalizados e inmunorreactivos para el fungicida fludioxonil

La presente invención se refiere a haptenos, conjugados, derivados marcados y anticuerpos para fludioxonil. Asímismo, la presente invención también se refiere al uso de conjugados de fludioxonil como antígenos de ensayo o inmunógenos para obtener anticuerpos de este fungicida; y al uso de los derivados marcados de fludioxonil como antígenos de ensayo. Además, la presente invención también se refiere a un método de análisis de fludioxonil utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcados. Esta invención también proporciona un kit para analizar fludioxonil que comprende anticuerpos de este fungicida, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcadosPeer reviewedConsejo Superior de Investigaciones Científicas, Universitat de ValènciaA1 Solicitud de patente con informe sobre el estado de la técnic

Fungicide multiresidue monitoring in international wines by immunoassays

24 pages, 5 figures, 1 table. -- Available online 10 November 2015.Azoxystrobin, boscalid, cyprodinil, fenhexamid, and pyrimethanil are new generation fungicides extensively employed in order to combat diseases affecting vineyards worldwide. Owing to their physico-chemical characteristics, residues of these compounds on grapes are transferred to must and wine. In this study, a survey of the occurrence of these fungicides in international wines was carried out by using rapid antibody-based assays. Results are discussed as a function of wine type and sample geographical origin. 44.4% of the samples contained at least one of the targets (>10 μg L−1). Fungicide residue occurrences were 22.4%, 19.2%, 18.8%, 6.8%, and 1.2% for pyrimethanil, boscalid, fenhexamid, cyprodinil, and azoxystrobin, respectively, while residue contents higher than 100 μg L−1 were found in 8.4% of the samples. This study shows that contamination of commercial wines with pesticides is an issue of worldwide relevance with potential implications for consumer health and international trade.This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2009-12940-C02-01-02/ALI and AGL2012-39965-C02-01-02/ALI) and cofinanced by FEDER funds.Peer reviewe

Ready Access to Proquinazid Haptens via Cross-Coupling Chemistry for Antibody Generation and Immunoassay Development

17 pages, 4 tables, 5 figures.-- Publisher's PDFBioconjugate preparation is a fundamental step for antibody generation and immunoassay development to small chemical compounds. For analytical targets holding in their structure an aryl halogen atom, cross-coupling reactions may be a simple and efficient way to obtain functionalized derivatives; thus offering great potential to elicit robust and selective immune responses after being coupled to immunogenic carrier proteins. However, substitution of the halogen atom by an aliphatic chain might eventually compromise the affinity and specificity of the resulting antibodies. In order to address this issue, proquinazid, a new-generation fungicide with outstanding performance, was chosen as model analyte. Two functionalized derivatives differing in spacer arm rigidity were synthesized by Sonogashira cross-coupling chemistry. These haptens were covalently coupled to bovine serum albumin and the resulting immunoconjugates were employed for rabbit vaccination. Antibodies were tested for proquinazid recognition by direct and indirect competitive immunoassay, and IC50 values in the low nanomolar range were found, thus demonstrating the suitability of this straightforward synthetic strategy for the generation of immunoreagents to compounds bearing an aryl halide. Following antibody characterization, competitive immunoassays were developed and employed to determine proquinazid residues in grape musts, and their analytical performance was satisfactorily validated by comparison with GC–MS. Besides having described the development of the first immunochemical method for proquinazid analysis, an efficient functionalization approach for analytes comprising aryl halides is reported.Peer reviewe

Monoclonal antibody-based immunoassays for cyprodinil residue analysis in QuEChERS-based fruit extracts

Cyprodinil is among the most common agrochemical residues found in highly perishable fruits, such as strawberries. In the present study, high-affinity monoclonal antibodies to this anilinopyrimidine fungicide were raised for the first time with the aim to produce valuable immunochemical analytical assays. Cyprodinil bioconjugates and the generated novel monoclonal antibodies were employed for sensitive competitive immunoassay development in two different formats. The limits of detection of the optimized assays were 20 and 30 ng L-1 for the indirect and direct assay, respectively. Influence over assay parameters of different physicochemical factors was studied. Strawberry samples were extracted following the recommended QuEChERS procedure for pesticide residues in food, and analyzed by the optimized immunoassays. Recoveries and coefficients of variation from fortified samples were within standard values. In addition, the obtained immunochemical results with naturally contaminated samples were statistically comparable, according to Deming regression analysis, to those of a reference chromatographic method.This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2009-12940-C02-01-02/ALI and AGL2012-39965-C02-01-02/ALI) and cofinanced by FEDER Funds. J.V.M. and F.A.E.-T. were hired by the Consejo Superior de Investigaciones Científicas (CSIC), the former under a Ramón y Cajal contract and the latter under a JAE-doc contract, both of them financed by Ministerio de Ciencia e Innovación and the European Social Fund – European Union. G.Q.-R. is a recipient of a PROMEP predoctoral fellowship from Secretaría de Educación Pública of the Mexican government. Proteomic analysis was carried out in the SCIE University of Valencia Proteomics Unit, a member of the ISCIII ProteoRedProteomics Platform.Peer Reviewe

Applications of quantum dots as probes in immunosensing of small-sized analytes

Quantum dots (QDs) are semiconductor nanoparticles with very interesting optical properties, like high quantum yield or narrow and size-tuneable fluorescence spectra. Current applications of QDs are widespread, their use as fluorescence labels in bioassays being one of the most promising. These nanoparticles are usually conjugated to highly specific biomolecules like antibodies, oligonucleotides, enzymes or aptamers to improve assay selectivity. In this review, QD surface passivation, conjugation to biomolecules, and purification strategies are discussed with special emphasis to the development of QD-based immunoassays for the detection of low molecular weight compounds given the relevance of this sort of analytes in health, food safety, pharmaceutical, or environmental monitoring areas. The aim of this review is to summarise the main achievements attained so far and to initialise researchers in the field of antibody-based assays employing QDs as labels, such as fluorescence-linked immunosorbent assay (FLISA), fluorescence (or Förster) resonance energy transfer (FRET), immunochromatographic methods, and immunosensors.This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2009-12940-C02-01-02/ALI) and cofinanced by FEDER funds. F.A.E.T. was hired by the Consejo Superior de Investigaciones CientÚficas (CSIC) under a JAE-doc contract financed by Ministerio de Ciencia e Innovación and the European Social Fund.Peer Reviewe