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[EN] The invention relates to haptenes, conjugates, marked derivates and antibodies for fludioxonil. The invention also relates to the use of conjugates of fludioxonil as test antigens or immunogens for producing antibodies of said fungicide; and to the use of the marked derivatives of fludioxonil as test antigens. The invention further relates to a method for analysing fludioxonil using the antibodies produced, sometimes together with test antigens that are conjugates or marked derivatives. The invention also provides a kit for analysing fludioxonil comprising antibodies of said fungicide, sometimes together with test antigens that are conjugates or marked derivatives, [ES] La presente invención se refiere a haptenos, conjugados, derivados marcados y anticuerpos para fludioxonil. Así mismo, 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 marcados

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 marcados

Trifloxystrobin is one of the main active principles belonging to the strobilurin family of crop protection compounds. In this article, the synthesis of a battery of regioisomeric functionalized derivatives of trifloxystrobin is described. The same aliphatic linear carboxylated chain was introduced as spacer arm in all of the synthesized haptens, but it was located at different positions of the parent molecule. N,N′-Disuccinimidyl carbonate was employed for hapten activation, so the resulting N-hydroxysuccinimyl ester could be readily purified and efficiently coupled to proteins. After immunization and hybridoma generation, a collection of 20 mouse monoclonal antibodies from different immunizing haptens was obtained. The analytical performance of these immunoreagents was evaluated in terms of affinity and selectivity with the aim to develop rapid and practical immunochemical procedures for trifloxystrobin determination. © 2013 Elsevier Ltd. All rights reserved.

Haptens, bioconjugates, and antibodies for highly sensitive immunochemical analysis of the new-generation fungicide penthiopyrad are described. Two haptens with equivalent carboxylated linkers were prepared, and the purified active esters were efficiently coupled to proteins. The results revealed slightly different antibody-eliciting capacities for the two synthetic derivatives. All of the produced antibodies were specific for penthiopyrad, and showed affinity values in the nanomolar range.

Kresoxim-methyl was one of the two first strobilurins to be discovered, and nowadays it is widely used as an antifungal agent in crop protection. Because of its low molecular weight and negligible structural complexity, the generation of antibodies to kresoxim-methyl noticeably requires the preparation of functionalized haptens. In this study, the introduction of a hydrocarbon spacer arm at the aromatic moieties of the target molecule was carried out by a convergent strategy based on the Sonogashira cross-coupling reaction, and functionalized linkers of the same length were also tethered to the aliphatic toxophore group by the O-alkylation reaction. Evaluation of the immune response, in terms of antibody affinity, showed a differential behavior among five synthesized haptens whose sole dissimilarity was the derivatization site. The characteristic (methoxyimino)acetate moiety of strobilurins was revealed as the optimum linker position for high-affinity polyclonal and monoclonal antibody production. However, good monoclonal antibodies were isolated from mice immunized with a hapten carrying the linker at an opposite site, which otherwise generated a poor polyclonal response in rabbits. Site-heterology was confirmed as a feasible approach for the improvement of the apparent affinity, particularly with polyclonal antibodies. Several of the monoclonal antibodies generated in the context of this project could be proper binders for kresoxim-methyl immunosensing over different analytical platforms.

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.

Rapid analytical methods enabling the determination of diverse targets are essential in a number of research areas, from clinical diagnostics to feed and food quality and safety. Herein, the development of a quantitative immunochromatographic assay for the detection of the synthetic phytoregulator forchlorfenuron (CPPU) is described. The competitive lateral flow immunoassay (LFIA) was based on the immobilization onto a nitrocellulose membrane of an ovalbumin-CPPU conjugate (test line) and on the use of an immunodetection ligand consisting of carbon nanoparticles labeled with an anti-CPPU monoclonal antibody through interaction with a secondary antibody. The presence of CPPU in horticultural samples was visually interpreted by the decrease in the black signal intensity of the test line, according to the competitive character of the format. The quantitative determination of the analyte was easily performed by a two-step procedure consisting of flatbed scanning of the strips followed by computer-based image analysis of the pixel gray volumes of the test lines. Under optimized conditions, the immunochromatographic test afforded a limit of quantification in buffer of 89. ng/L. The accuracy of the strip test was assessed by the analysis of fruit samples with incurred residues, and the obtained results were compared with those derived from two reference methods, ELISA and HPLC. The LOQ of the CPPU-specific LFIA in kiwifruits and grapes was established at 33.4 οg/kg. The excellent analytical performance of the developed strip test demonstrates the potential of immunochromatographic assays for the quantitative monitoring of small organic molecules in complex matrices. © 2012 Elsevier B.V.

Mepanipyrim is an anilinopyrimidine fungicide used worldwide for crop protection. With the aim of developing useful immunoreagents for mepanipyrim immunoanalysis, two new functionalized derivatives were prepared and antibodies were generated. Affinity and specificity were assessed by direct and indirect competitive ELISA using homologous and heterologous conjugates. Although all antibodies were selective for the target analyte, the immunizing hapten structure was revealed as a determinant for high-affinity antibody production (IC50 = 3 nM). © 2013 The Royal Society of Chemistry.

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.

La presente invención se refiere a haptenos, conjugados, derivados marcados y anticuerpos para ciprodinil. Así mismo, la presente invención también se refiere al uso de conjugados de ciprodinil como antígenos de ensayo o inmunógenos para obtener anticuerpos de este fungicida; y al uso de los derivados marcados de ciprodinil como antígenos de ensayo. Además, la presente invención también se refiere a un método de análisis de ciprodinil 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 ciprodinil que comprende anticuerpos de este fungicida, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcados.

La presente invención se refiere a haptenos, conjugados, derivados marcados y anticuerpos para pirimetanil. Así mismo, la presente invención también se refiere al uso de conjugados de pirimetanil como antígenos de ensayo o inmunógenos para obtener anticuerpos de este fungicida; y al uso de los derivados marcados de pirimetanil como antígenos de ensayo. Además, la presente invención también se refiere a un método de análisis de pirimetanil 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 pirimetanil que comprende anticuerpos de este fungicida, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcados.

[EN] The invention relates to haptens, conjugates and antibodies for pyrimethanil fungicide. In addition, the invention relates to the use of pyrimethanil conjugates as assay antigens or immunogens in order to obtain antibodies of the aforementioned fungicide, and to the use of the labelled derivatives of pyrimethanil as assay antigens. The invention also relates to a pyrimethanil analysis method using the antibodies obtained, at times together with assay antigens which are conjugates or labelled derivatives. The invention also provides a kit for analysing pyrimethanil, which includes antibodies of said fungicide, at times together with assay antigens which are conjugates or labelled derivatives., [ES] La presente invención se refiere a haptenos, conjugados, derivados marcados y anticuerpos para pirimetanil. Así mismo, la presente invención también se refiere al uso de los derivados marcados de pirimetanil como antígenos de ensayo o inmunógenos para obtener anticuerpos de este fungicida; y al uso de los derivados marcados de pirimetanil como antígenos de ensayo. Además, la presente invención también se refiere a un método de análisis de pirimetanil 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 pirimetanil que comprende anticuerpos de este fungicida, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcados.

[EN] The invention relates to haptens, conjugates, labelled derivatives and antibodies for cyprodinil. Likewise, the present invention also relates to the use of cyprodinil conjugates as test antigens or immunogens for obtaining antibodies of said fungicide, and to the use of the labelled derivatives of cyprodinil as test antigens. Furthermore, the present invention also relates to a method for analysing cyprodinil using the thus obtained antibodies, at times together with the test antigens, which are conjugates or labelled derivatives. The invention also provides a kit for analysing cyprodinil, which includes antibodies of said fungicide, at times together with test antigens, which are conjugates or labelled derivatives., [ES] La presente invención se refiere a haptenos, conjugados, derivados marcados y anticuerpos para cipronil. Así mismo, la presente invención también se refiere al uso de conjugados de ciprodinil como antigenos de ensayo o inmunógenos para obtener anticuerpos de este fungicida; y al uso de los derivados marcados de ciprodinil como antigenos de ensayo. Además, la presente inveción también se refiere a un método de análisis de ciprodinil utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcados. Esta invención también se proporciona un kit par analizar ciprodinil que comprende anticuerpos de este fungicida, en ocasiones junto con antígenos de ensayo que son conjugados o derivados marcados.

Cyprodinil is an anilinopyrimidine fungicide applied worldwide for the prevention and treatment of highly destructive plant diseases in a large variety of crops, including cereals, fruits, and vegetables. This paper describes the development of the first reported immunoassays for cyprodinil. Two original haptens have been synthesized and conjugated to different carrier proteins, and polyclonal antibodies have been produced. Moreover, competitive enzyme-linked immunosorbent assays have been developed and characterized for the analysis of this widely used pesticide. The influence of organic solvents and buffer conditions over the assay analytical parameters was studied. The IC 50 values of the optimized immunoassays were 1.6 and 2.8 ng/mL for the direct and indirect formats, respectively. Quantitative recoveries were found using spiked apple and grape juice samples after a simple direct dilution, and a limit of quantification of 20 ng/mL for both fruit matrices was achieved. These immunoreagents could be very valuable for the sensitive, straightforward, and rapid monitoring of cyprodinil residues in foodstuffs.

[EN] The present invention relates to novel chemically functionalized boscalid derivatives. Furtherrnore, the present invention relates to boscalid irnmuno gens , to the conjugated or labelled derivatives and to the antibodies obtained from the irnmunogens of the present invention., [ES] La presente invención derivados se refiere a nuevos funcionalizados químicamente de boscalid. Además la presente invención se refiere a inmunogenos de boscalid, a los derivados conjugados o marcados y a los anticuerpos que se obtienen a partir de los inmunogenos de la presente invención.

La presente invención se refiere a nuevos derivados funcionalizados químicamente de boscalid. Además la presente invención se refiere a inmunógenos de boscalid, a los derivados conjugados o marcados y a los anticuerpos que se obtienen a partir de los inmunógenos de la presente invención.

The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate.

To obtain highly-specific and selective forchlorfenuron binders, a collection of functionalized derivatives with different spacer arm locations and lengths was prepared. By immunization with target-mimicking haptens, a large battery of monoclonal and polyclonal antibodies against this synthetic cell regulator was produced and exhaustively characterized in two immunoassay formats using homologous and heterologous conjugates. Antibodies with IC50 values lower than 0.3 nM were successfully raised from the prepared immunogens, thus evidencing the efficacy of the explored strategies. In order to identify significant epitopes in the antibody-antigen interaction, a series of new chemical forchlorfenuron analogues, with slight modifications at both rings of the target molecule, were synthesized and evaluated in competitive assays. As a novel approach in hapten recognition studies, data processing was performed by computational classification methods based on hierarchical clustering. This strategy was shown to be highly valuable for a straightforward profiling of antibodies according to analogue recognition patterns. A relationship could be established between the antigen binding properties of antibodies and the structure of the immunogen. Whereas antibodies with equivalent affinities had been obtained from all of the derivatives, their specificity was found to be largely influenced by the differential exposition of the molecule to the immune system.

El objetivo de esta tesis fue desarrollar un ELISA multianalito, basado en anticuerpos monoclonales, para los plaguicidas N-metilcarbamatos carbaryl, carbofuran y methiocarb. Para ello se desarrollaron en primer lugar ELISAs individuales para carbofuran y methiocarb. Posteriormente estos inmunoensayos, junto con el de carbary1, previamente desarrollado en el LIB, se integraron en un ELISA multianalito. Para producir anticuerpos monoclonales frente a carbofuran y methiocarb se sintetizaron tres compuestos para cada analito, con un brazo espaciador carboxílico de diferente longitud introducido en el grupo carbamato de la estructura del analito. Posteriormente, estos compuestos fueron conjugados a proteínas y utilizados para inmunizar ratones. Los anticuerpos monoclonales producidos fueron caracterizados en los formatos de ELISA de conjugado inmovilizado (C1) y anticuerpo inmovilizado (Al), usando otros compuestos sintetizados como haptenos heterólogos de ensayo. Los anticuerpos de methiocarb LIB-MXNB31 y de carbofuran LIB-BFNB67, en combinación con los haptenos de methiocarb (DPNI-1 y MCNH) y de carbofuran (BFNH), acoplados a ovoalbúmina y a peroxidasa, respectivamente, se utilizaron para desarrollar ELISAs en los formatos Cl y Al, respectivamente. Los inmunoensayos desarrollados para carbofuran y methiocarb mostraron valores de I5o aproximadamente de 0.7 y 0.03 ng/ml, respectivamente. Los ELISAs de methiocarb fueron muy específicos, mientras que los de carbofuran reconocieron algunos compuestos estructuralmente similares. Los inmunoensayos de carbofuran y methiocarb, junto con el de carbary1, fueron validados frente a HPLC, como método de referencia, en el análisis de muestras purificadas de frutas y hortalizas, obteniéndose una buena correlación entre métodos. Asimismo, se demostró que estos ELISAs puede ser aplicados al análisis de carbary1, carbofuran y methiocarb en extractos crudos, obteniéndose incluso mejores resultados que en las muestras purificadas.........

[EN] Immunosensor systems have been developed for the rapid and sensitive determination of pesticides, using the insecticide carbaryl as a model analyte. The systems are based on the principle of heterogeneous competitive enzyme immunoassay and used mouse monoclonal anti-carbaryl antibodies either in solution (indirect format) or immobilized (direct format). In both formats, enzyme label (horseradish peroxidase, HRP) and fluorometric detection were employed. In the direct format, antibodies were immobilized on a hydrazide-derivatized agarose gel, and 6-[((1-naphthyloxy)carbonyl)amino]hexanoic acid (CNH hapten) conjugated to HRP was used as the enzyme tracer. The limit of detection was 26 ng L-1 (11 min/assay), and the useful life of the sensor was 60−70 cycles. In the indirect format, CNH conjugated to bovine serum albumin was immobilized on an N-hydroxysuccinimide-derivatized agarose gel, and a rabbit anti-mouse antibody labeled with HRP was used as a secondary immunoreagent. The limit of detection was 284 ng L-1 (17 min/assay), and the useful life of the sensor was 160−200 cycles. The developed methods were applied to the analysis of commercial drinking water and apple juice spiked with carbaryl. Interassay RSD ranged from 9 to 39% in the direct format and from 4 to 28% in the indirect format. Recoveries were between 62 and 109% in the direct format and between 78 and 124% in the indirect format. The results were compared with those obtained by enzyme-linked immunosorbent assay as reference method and indicated the suitability of the immunosensor for quality control in water and food analysis

[EN] The application of controlled-pore glass (CPG) as solid support for immobilization of immunoreagents in order to develop flow-through immunosensors is described. Monoclonal antibodies (MAbs) to carbaryl were site-directed immobilized on CPG through covalent attachment of their oxidized carbohydrate moieties to amine groups generated on the surface of silanyzed CPG. The automated immunosensor system is based on the LIB-CNA36 MAb in a direct competitive assay format, with horseradish peroxidase as enzyme label and fluorimetric detection. The dynamic range of the sensor is 0.05-1 mu g l(-1), with a detection limit of 0.029 mu g l(-1), being sensitive enough to be applied to drinking water samples without preconcentration. The immobilized antibody reactor is able to run a whole assay in 20 min, and is reusable for more than 100 of consecutive assay cycles without significant loss of performance. The recognition of 1-naphthol -the main metabolite of carbaryl -and other N-methylcarbamate insecticides are also studied, none of these compounds showing cross-reactivity higher than 7%. A preliminary validation of the immunosensor, carried out by analysing real samples spiked with carbaryl, shows good results for bottled water and for commercial honey diluted with PBS (1 g l(-1)) as the only sample pretreatment.

[EN] Monoclonal antibodies (MAbs) for the carbamate insecticide carbaryl (1-naphthyl N-methylcarbamate) were obtained by immunizing mice with 6-[[(1-naphthyloxy)carbonyl]amino]hexanoic acid (3, CNH) conjugated to bovine serum albumin (BSA). The hapten synthesis strategy was designed to preserve the carbamate group characteristic of N-methylcarbamate pesticides. The most promising MAb (LIB/CNH-3.6) was characterized by using an indirect homologous antigen-coated ELISA. With this assay format, LIB/CNH-3.6 showed a high affinity for carbaryl (I-50 = 3 6 nM) and minimum cross-reactivities for several N-methylcarbamates and 1-naphthol, the main carbaryl metabolite. Both affinity and specificity achieved by LIB/CNH-3.6 meant a great improvement with respect to the features displayed by the serum of the mouse from which the MAb was derived. This MAb appears to be a very promising immunoreagent for the future development of a specific and sensitive quantitative ELISA for carbaryl, and the applied hapten synthesis strategy is proposed as a general procedure to obtain immunogenic conjugates for other N-methylcarbamate pesticides.

[EN] A competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies was developed to measure nicotine in smoke condensates of cigarettes. The ELISA standard curve displays a detection limit of 70 ng/mL and an effective working range of 0.3-4.0 mug of nicotine/mL. Using standard samples, ELISA results compare well with theoretical values (r = 0.997). The suitability of the method for industrial routine application was evaluated by measuring the nicotine concentration in 245 cigarette smoke condensates. When ELISA results were compared with those obtained by gas chromatography, a correlation coefficient r = 0.886 was obtained. The assay allows the specific, precise, and accurate determination of nicotine, without any significant interference from other alkaloids also present in tobacco smoke. Preliminary data suggest that the immunoassay could also be applied to other matrices, e.g., tobacco extracts. ELISA equipment requirements are minimum, simple, and low-cost, and an unskilled person could perform 150 analysis in a working day. On the basis of these features, ELISA is proposed as a promising alternative to instrumental methods for some industrial applications.