Search

Alternariol is an emerging mycotoxin that may be present in many foods at relatively high concentrations. Therefore, sensitive analytical methods are needed to reduce human exposure to this secondary fungal metabolite. The aim of the present study was to develop alternative immunochemical methods for the rapid analysis of alternariol using novel homemade monoclonal antibodies. In this study, a direct competitive enzyme-linked immunosorbent assay for alternariol is reported for the first time, with an IC50 value and a detection limit of 0.020 ng/mL and 0.004 ng/mL, respectively. Under the optimized immunoassay conditions, alternariol could be accurately and precisely quantified in fruit juice samples in only 25 min. In addition, a nanoparticle-based lateral-flow immunochromatography assay with an IC50 value and a visual detection limit of 0.24 ng/mL and 1 ng/mL, respectively, was developed. This immunoassay was validated according to European guidelines for portable semiquantitative analytical methods. Thus, juice samples with alternariol levels above 4 ng/mL were detected by this rapid method in only 15 min, with a negligible false suspect rate.

Alternariol is a toxic secondary metabolite produced by Alternaria fungi. Nowadays, this mycotoxin can be found in many products of plant origin at concerning concentrations. The aim of the present study was to develop a highly sensitive and selective immunochemical method for the analysis of alternariol. To this end, hapten synthesis was carried out from the scratch and specific high-affinity monoclonal antibodies to alternariol were generated by using, for the first time, alternariol bioconjugates with unambiguous linker tethering sites. A novel indirect enzyme-linked immunosorbent assay that incorporated a rationally designed heterologous conjugate was developed. The optimized assay showed an outstanding half-maximal inhibition concentration for alternariol below 0.04 ng/mL, and no cross-reactivity with alternariol monomethyl ether was observed. Very good recovery values and coefficients of variation were obtained from the analysis of alternariol-fortified fruit and cereal flour samples. Finally, alternariol was quantitatively determined in pears that had been previously infected with Alternaria alternata, showing excellent correlation with liquid chromatography results.

Highly active antiretroviral therapy (HAART) includes very potent drugs that are often characterized by high toxicity. Tenofovir (TFV) is a widely used drug prescribed mainly for pre-exposure prophylaxis (PreP) and the treatment of human immunodeficiency virus (HIV). The therapeutic range of TFV is narrow, and adverse effects occur with both underdose and overdose. The main factor contributing to therapeutic failure is the improper management of TFV, which may be caused by low compliance or patient variability. An important tool to prevent inappropriate administration is therapeutic drug monitoring (TDM) of compliance-relevant concentrations (ARCs) of TFV. TDM is performed routinely using time-consuming and expensive chromatographic methods coupled with mass spectrometry. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs), are based on antibody–antigen specific recognition and represent key tools for real-time quantitative and qualitative screening for point-of-care testing (POCT). Since saliva is a non-invasive and non-infectious biological sample, it is well-suited for TDM. However, saliva is expected to have a very low ARC for TFV, so tests with high sensitivity are required. Here, we have developed and validated a highly sensitive ELISA (IC50 1.2 ng/mL, dynamic range 0.4–10 ng/mL) that allows the quantification of TFV in saliva at ARCs and an extremely sensitive LFIA (visual LOD 0.5 ng/mL) that is able to distinguish between optimal and suboptimal ARCs of TFV in untreated saliva.

The present invention relates to bioconjugates and labeled derivatives of the adducts formed by the reaction of patulin with benzene-1,2-dithiols or their salts. Said conjugates are suitable for the production of antibodies capable of recognizing the aforementioned patulin-(benzene-1,2-dithiols) adducts with high affinity and specificity. Likewise, the present invention also relates to the use of bioconjugates of patulin-(benzene-1,2-dithiols) adducts and of labeled derivatives thereof as assay antigens. Furthermore, the present invention also relates to immunochemical methods for the analysis of patulin, using the bioconjugates and labeled derivatives and antibodies of the invention, which include a previous step to quantitatively convert patulin into these adducts. This invention also provides a kit for analyzing patulin comprising a benzene-1,2-dithiol or its salts, antibodies against patulin-(benzene-1,2-dithiols) adducts, and assay antigens.

Weed resistance to glyphosate has been a driving force behind the increased use of alternative herbicides in agriculture. Recently, dicamba-tolerant recombinant plants were introduced to the market, which may result in residues of this agrochemical contaminating environmental waters. Given that restrictions on the use of dicamba have consequently been established by regulatory agencies, it is therefore also desirable to conduct extensive controls on dicamba residues. Immunoassays are currently the most powerful bioanalytical technology for the rapid monitoring of chemical residues and contaminants. In the present study, a novel hapten was designed maintaining unaltered all the antigenic moieties of the target molecule, and this was used to generate high-affinity monoclonal antibodies against dicamba for the first time. Additionally, a collection of haptens with different linker composition or linker tethering site was synthesized and conjugated to proteins. Using these novel immunoreagents, a direct competitive enzyme-linked immunosorbent assay with a limit of detection for dicamba of 0.24 ng/mL was developed and validated. Analysis of water samples from different origins afforded recovery values between 90 % and 120 %, and coefficients of variation below 20 % were obtained. These results indicate that the developed immunochemical assay is suitable for the rapid determination of dicamba residues in environmental water samples.

Algal blooms that contaminate freshwater resources with cyanotoxins constitute, nowadays, a global concern. To deal with this problem, a variety of analytical methods, including immunochemical assays, are available for the main algal toxins, for example, microcystins, nodularins, and saxitoxins, with the remarkable exception of anatoxin-a. Now, for the first time, highly sensitive, enantioselective immunoassays for anatoxin-a have been validated using homemade monoclonal antibodies. Two competitive enzyme-linked immunosorbent assays were developed in different formats, with detection limits for (+)-anatoxin-a of 0.1 ng/mL. Excellent recovery values between 82 and 117%, and coefficients of variation below 20%, were observed using environmental water samples fortified between 0.5 and 500 ng/mL. In addition, a lateral-flow immunochromatographic assay was optimized for visual and instrumental reading of results. This test showed a visual detection limit for (+)-anatoxin-a of 4 ng/mL. Performance with a reader was validated in accordance with the European guidelines for semiquantitative rapid methods for small chemical contaminants. Thus, at a screening target concentration of 2 ng/mL, the probability of a blank sample to be classified as "suspect" was as low as 0.2%. Finally, the optimized direct enzyme immunoassay was validated by comparison with high-performance liquid chromatography-tandem mass spectroscopy data and showed a good correlation (r = 0.995) with a slope of 0.94. Moreover, environmental water samples containing more than 2 ng/mL of anatoxin-a were detected by the developed dipstick assay. These results provide supplementary and complementary strategies for monitoring the presence of anatoxin-a in water.

Appropriate hapten design and synthesis have been identified as critical steps to gener-ate 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.

La presente invención se refiere a bioconjugados y derivados marcados de los aductos formados por la reacción de patulina con benceno-1,2-ditioles o sus sales. Dichos conjugados resultan adecuados para la producción de anticuerpos capaces de reconocer con elevada afinidad y especificidad los mencionados aductos patulina-(benceno-1,2-ditioles). Asimismo, la presente invención también se refiere al uso de bioconjugados dé los aductos patulina-(benceno-1,2-ditioles) y de derivados marcados de los mismos como antigenos de ensayo. Además, la presente invención también se refiere a métodos inmunoquímicos de análisis de patulina, utilizando los bioconjugados y derivados marcados y anticuerpos de la invención, que incluyen un paso previo para convertir cuantitativamente la patulina en estos aductos. Esta invención también proporciona un kit para analizar patulina que comprende un benceno-1,2-ditiol o sus sales, anticuerpos frente a los aductos patulina--(benceno-1,2-ditioles), y antígenos de ensayo. [ES], The present invention relates to bioconjugates and labeled derivatives of the adducts formed by the reaction of patulin with benzene-1,2-dithiols or their salts. Said conjugates are suitable for the production of antibodies capable of recognizing the aforementioned patulin-(benzene-1,2-dithiol) adducts with high affinity and specificity. Likewise, the present invention also relates to the use of bioconjugates of patulin-(benzene-1,2- dithiol) adducts and of labeled derivatives thereof as assay antigens. Furthermore, the present invention also relates to immunochemical methods for the analysis of patulin, using the bioconjugates and labeled derivatives and antibodies of the invention, which include a previous step to convert patulin quantitatively into these adducts. This invention also provides a kit for analyzing patulin comprising a benzene-1,2-dithiol or its salts, antibodies against patulin-(benzene-1,2-dithiol) adducts, and assay antigens. [EN]

Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and agrochemical samples. A thorough characterization of the antibody‒antigen interaction can bring light to the immunoreagent selection process in order to develop sensitive and robust tests. Thus, determination of equilibrium and reaction rate constants is usually recommendable. However, this can be quite tricky for low molecular weight compounds, and competitive strategies are commonly followed to estimate apparent affinity values. In the present study, a collection of monoclonal antibodies to penthiopyrad was raised for the first time, and apparent equilibrium constants were assessed by the Langmuir model using three different competitive enzyme-linked immunosorbent assay formats. The obtained KD values from antibody-coated assays were quite close to the corresponding KD values calculated from surface plasmon resonance (SPR) evaluation. These studies were employed to select a pair of immunoreagents for immunoassay development. The KD value for penthiopyrad of the selected antibody obtained by SPR was 0.28 nM. The optimized direct assay showed an IC50 value for penthiopyrad of 0.42 nM (0.15 ng mL−1) in buffer. The limit of quantification for grape, must, and wine samples was 10 ng mL−1. An excellent correlation was found when immunochemical results were compared with those from LC-MS/MS. As an application case, it was determined that 58% of penthiopyrad was still found in wine after fermentation.

Immunochemical methods for mycotoxin analysis require antigens with well-defined structures and antibodies with outstanding binding properties. Immunoreagents for the mycotoxins alternariol and/or alternariol monomethyl ether have typically been obtained with chemically uncharacterized haptens, and antigen conjugates have most likely been prepared with mixtures of functionalized molecules. For the first time, total synthesis was performed, in the present study, to obtain two haptens with opposite linker attachment locations. The functionalized synthetic haptens were purified and deeply characterized by different spectrometric methods, allowing the preparation of bioconjugates with unequivocal structures. Direct and indirect competitive enzyme-linked immunosorbent assays, using homologous and heterologous conjugates, were employed to extensively evaluate the generated immunoreagents. Antibodies with high affinity were raised from conjugates of both haptens, and a structure-activity relationship between the synthetic haptens and the specificity of the generated antibodies could be established. These results pave the way for the development of novel highly sensitive immunoassays selective of one or two of these Alternaria mycotoxins.

Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin.

[ES] La presente invención se refiere a bioconjugados de análogos fragmentarios de aflatoxinas que tienen propiedades novedosas hacia los anticuerpos producidos frente a bioconjugados de aflatoxina M1. Asimismo, la presente invención también se refiere al uso de estos bioconjugados y derivados marcados de estos análogos fragmentarios como antígenos de ensayo. Además, la presente invención también se refiere a métodos de análisis utilizando estos bioconjugados o derivados marcados junto a anticuerpos producidos frente a aflatoxina M1. Esta invención también proporciona un kit para analizar aflatoxina M1 que comprende conjugados o derivados marcados de estos derivados fragmentarios junto con anticuerpos producidos frente a aflatoxina M1., [EN] The present invention relates to bioconjugates of fragmentary analogues of aflatoxins having novel properties towards antibodies produced against Aflatoxin bioconjugates M1. Likewise, the present invention also relates to the use of these bioconjugates and marked derivatives of these fragmentary analogues such as test antigens. In addition, the present invention also relates to methods of analysis using these bioconjugates or derivatives marked together with antibodies produced against aflatoxin M1. This invention also provides a kit for analyzing M1 aflatoxin comprising conjugates or marked derivatives of these fragmentary derivatives together with antibodies produced against aflatoxin M1.

Immunochemical methods are highly deployed in analytical laboratories worldwide for monitoring the incidence of mycotoxins in the food chain. Nevertheless, most conventional immunoassays for ochratoxin A (OTA), including commercial kits, show limitations to robustly determine this mycotoxin in grape-derived products below regulated levels (2 ng/mL). Herein, two rapid tests for sensitive OTA determination in wine and must were developed capitalizing on a collection of bioconjugates from innovative synthetic haptens and monoclonal antibodies with subnanomolar affinity. The ELISA (LOD = 8 pg/mL) showed excellent performance in recovery studies, and it was applied to survey commercial wines and musts for OTA contamination. Concerning LFIA, validation according to the Commission Regulation 519/2014 showed that samples exceeding 2 ng/mL were properly scored as uncompliant. More importantly, illegal samples provided a complete inhibition of the test signal, making this test an easy-to-use, rapid, and convenient screening method for in-house control of OTA in wineries.

Approximately 32 million people have died of HIV infection since the beginning of the outbreak, and 38 million are currently infected. Among strategies adopted by the Joint United Nations Programme on HIV/AIDS to end the AIDS global epidemic, the treatment, diagnosis, and viral suppression of the infected subjects are considered crucial for HIV prevention and transmission. Although several antiretroviral (ARV) drugs are successfully used to manage HIV infection, their efficacy strictly relies on perfect adherence to the therapy, which is seldom achieved. Patient supervision, especially in HIV-endemic, low-resource settings, requires rapid, easy-to-use, and affordable analytical tools, such as the enzyme-linked immunosorbent assay (ELISA) and especially the lateral flow immunoassay (LFIA). In this work, high-affinity monoclonal antibodies were generated to develop ELISA and LFIA prototypes for monitoring tenofovir (TFV), an ARV drug present in several HIV treatments. TFV was functionalized by inserting a carboxylated C5-linker at the phosphonic group of the molecule, and the synthetic derivative was conjugated to proteins for mice immunization. Through a rigorous screening strategy of hybridoma supernatants, a panel of monoclonal antibodies strongly binding to TFV was obtained. Following antibody characterization for affinity and selectivity by competitive ELISA, a LFIA prototype was developed and tentatively applied to determine TFV in simulated urine. The point-of-care test showed ultra-high detectability (the visual limit of detection was 2.5 nM, 1.4 ng mL−1), excellent selectivity, and limited proneness to matrix interference, thus potentially making this rapid method a valuable tool for the on-site assessment of patient adherence to ARV therapy.

[ES] El control efectivo de la calidad y seguridad de los ali­mentos requiere de métodos analíticos que garanticen la deter­minación fiable de cualquier sustancia potencialmente perjudicial para el consumidor que pueda estar presente en el alimento antes de su distribución y comercialización. Una de las aproximaciones analíticas que contribuye a garantizar este objetivo engloba una serie de técnicas que tienen en común la utilización de anticuerpos como elementos esenciales para la detección del analito diana, y que en conjunto reciben el nombre de métodos inmunoquímicos. Este artículo pretender aportar una visión básica de los principios bioquímicos subyacentes a estas tecnologías y cuáles son sus venta­jas y limitaciones en la determinación de contaminantes químicos, residuos y aditivos en matrices alimentarias. En la última parte se comentan algunas de nuestras iniciativas en este ámbito que han dado lugar a kits rápidos disponibles comercialmente tras haber transferido la tecnología correspondiente al sector industrial.

Bioorthogonal reactions have revolutionized the way low molecular weight compounds are coupled to biomolecules. Organic chemistry, polymer science, and chemical biology are among the disciplines that are benefited the most from this breakthrough. Despite the reliability of the click chemistry concept for the efficient and chemoselective functionalization of biomacromolecules with haptens at preferred positions, the fact that azide–alkyne cycloaddition reactions originate new chemical moieties as part of the linker may have delayed their application in the immunodiagnostic field. Using the mycotoxin ochratoxin A as a model compound, we herein demonstrate for the first time that bioconjugates arising from the ligation between an azido-bearing hapten and an alkyne-modified carrier protein are able to elicit the generation of high-affinity monoclonal antibodies suitable for the development of rapid methods for the immunodetection of small organic molecules.

A direct competitive immunosensor for the electrochemical determination of Imidacloprid (IMD) pesticide on gold nanoparticle-modified screen-printed carbon electrodes (AuNP-SPCE) is here reported for the first time. Self-obtained specific monoclonal antibodies are immobilized on the AuNP-SPCE taking advantage of the AuNPs biofunctionalization abilities. In our biosensor design, free IMD in the sample competes with IMD conjugated with horseradish peroxidase (IMD-HRP) for the recognition by the antibodies. After that, 3,3′,5,5′-Tetramethylbenzidine (TMB) is enzymatically oxidized by HRP, followed by the oxidized TMB reduction back at the surface of the SPCE. This process gives an associated catalytic current (analytical signal) that is inversely proportional to the IMD amount. The main parameters affecting the analytical signal have been optimized, reaching a good precision (repeatability with a RSD of 6%), accuracy (relative error of 6%), stability (up to one month), selectivity and an excellent limit of detection (LOD of 22 pmol L−1), below the maximum levels allowed by the legislation, with a wide response range (50–10000 pmol L−1). The detection through antibodies also allows to have an excellent selectivity against other pesticides potentially present in real samples. Low matrix effects were found when analysing IMD in tap water and watermelon samples. The electrochemical immunosensor was also validated with HPLC-MS/MS, the reference method used in official laboratories for IMD analysis, through statistical tests. Our findings make the electrochemical immunosensor as an outstanding method for the rapid and sensitive determination of IMD at the point-of-use.

Monoclonal antibody-based techniques have become a useful analytical technology in the agro-food sector. Nowadays, residues of the recently registered fungicide fluopyram are increasingly being found in quality control programs. In the present study, novel chemical derivatives of this pesticide were prepared and specific and high-affinity monoclonal antibodies to fluopyram were raised for the first time. Moreover, immunoassays to fluopyram were developed in two alternative enzyme-linked immunosorbent assay formats, using homologous and heterologous assay conjugates, with limits of detection below 0.05 µg L−1. The optimized immunoassays were applied to the analysis of fluopyram in fortified plums and grapes of four different varieties as well as in in-house prepared musts and wines. Recoveries were between 76.3% and 109.6% and coefficients of variation were below 20%. Quantification limits were well below the maximum residue limits. Immunoassay performance was statistically validated with a reference chromatographic technique using samples from fluopyram-treated plum and grape cultivars.

La presente invención se refiere a bioconjugados y derivados marcados de ocratoxina A por posiciones diferentes de la molécula, adecuados para la producción de anticuerpos de elevada afinidad para ocratoxina A. Asimismo, la presente invención también se refiere al uso de bioconjugados de ocratoxina A y de derivados marcados de ocratoxina A como antígenos de ensayo. Además, la presente invención también se refiere a métodos de análisis, concentración y extracción de ocratoxina A utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son bioconjugados o derivados marcados. Esta invención también proporciona un kit para analizar ocratoxina A que comprende anticuerpos frente a esta micotoxina, en ocasiones junto con antígenos de ensayo que son bioconjugados o derivados de la ocratoxina A

The present invention relates to bioconjugates and derivatives of ochratoxin A labelled at different positions in the molecule suitable for the production of antibodies with a high affinity for ochratoxin A. Moreover, the present invention also relates to the use of ochratoxin A bioconjugates and labelled ochratoxin A derivatives as test antigens. The present invention also relates to methods for the analysis, concentration and extraction of ochratoxin A using the antibodies obtained, together with test antigens that are bioconjugates or labelled derivatives. This invention also provides a kit for analysing ochratoxin A comprising antibodies against said mycotoxin, in certain cases together with test antigens that are bioconjugates or labelled derivatives of ochratoxin A. [EN], La presente invención se refiere a bioconjugados y derivados marcados de ocratoxina A por posiciones diferentes de la molécula, adecuados para la producción de anticuerpos de elevada afinidad para ocratoxina A. Asimismo, la presente invención también se refiere al uso de bioconjugados de ocratoxina A y de derivados marcados de ocratoxina-A como antígenos de ensayo. Además, la presente invención también se refiere a métodos de análisis, concentración y extracción de ocratoxina A utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son bioconjugados o derivados marcados. Esta invención también proporciona un kit para analizar ocratoxina A que comprende anticuerpos frente a esta micotoxina, en ocasiones junto con antígenos de ensayo que son bioconjugados o derivados marcados de la ocratoxina A. [ES]

Spirotetramat - a tetramic acid insecticide - is rapidly metabolized or degraded to give spirotetramat-enol; so, common residue definitions include the sum of both compounds. In the present study, two spirotetramat-functionalized derivatives (haptens) have been designed to generate immunoreagents to these molecules for rapid immunochemical analysis. Haptens have been synthesized with alternative linker tethering sites and, for the first time, high-affinity antibodies have been generated with different specificities to these active principles. Two sensitive assays have been developed using the same antibody in different formats, and by using linker-site heterologous haptens, the selectivity of the final immunoassay could be improved. A generic immunoassay with sensitivity similar to spirotetramat and spirotetramat-enol and a specific assay of spirotetramat-enol have been developed. The described antibody and bioconjugates showed great potential for sensitive immunosensor development and analysis of this complex analyte.

Ochratoxin A is a potent toxic fungal metabolite whose undesirable presence in food commodities constitutes a problem of public health, so it is strictly regulated and controlled. For the first time, two derivatives of ochratoxin A (OTAb and OTAd) functionalized through positions other than the native carboxyl group of the mycotoxin, have been synthesized in order to better mimic, during the immunization process, the steric and conformational properties of the target analyte. Additionally, two conventional haptens making use of that native carboxyl group for protein coupling (OTAe and OTAf) were also prepared as controls for the purpose of comparison. The immunological performance in rabbits of protein conjugates based on OTAb and OTAd overcome that of conjugates employing OTAe and OTAf as haptens. After immunization of mice with OTAb and OTAd conjugates, a collection of high-affinity monoclonal antibodies to ochratoxin A was generated. In particular, one of those antibodies, the so-called OTAb#311, is very likely the best antibody produced so far in terms of selectivity and affinity to ochratoxin A.

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.

Immunochemical methods have been consolidated during the last few years as complementary analytical strategies for chemical contaminant and residue determination. However, generation of suitable immunoreagents for small organic molecules demands adequate hapten design. In this study, fenhexamid was considered as a model compound and novel haptens were designed and synthesized in order to evaluate the influence of the linker tethering site on antibody binding properties and immunoassay parameters. Haptens were conceived with the spacer arm at different positions, while the more antigenic aromatic moiety was kept free. The synthesis of these functionalized compounds was accomplished by total construction of the molecule through several steps. This strategy afforded very high-affinity monoclonal antibodies specific of fenhexamid, with IC50 values around or below 0.1 nM. Using these novel immunoreagents, a direct competitive enzyme-linked immunosorbent assay with a remarkably low limit of detection (4 ng L−1) was developed for the determination of fenhexamid residues. The selected immunoassay was investigated in terms of trueness, precision, repeatability, and robustness. The QuEChERS extraction methodology was applied to fortified samples and recoveries between 83% and 113%, with relative standard deviations below 20%, were observed. Moreover, contaminated and blind spiked samples were measured by the developed immunoassay and by ultra-performance liquid chromatography coupled to tandem mass spectrometry, showing statistically comparable results.

Boscalid is an agrochemical recently developed for crop protection and the most significant member of the succinate dehydrogenase inhibitor group of fungicides. In this study, a collection of high-affinity monoclonal antibodies was generated to boscalid. By using a series of haptens with a linker at alternative tethering sites of the boscalid framework, specific antibodies were isolated as well as antibodies that also recognized the main boscalid metabolite. Two immunoassays were developed using different ELISA formats. Optimized assays displayed very high sensitivities (limits of detection were near 0.01 µg/L). Trueness and precision for the determination of the target analyte in strawberry samples was evaluated. Moreover, immunoassay performance was validated with a reference chromatographic method using QuEChERS extracts of fruits from fungicide-treated crops. A monitoring study with strawberry samples from local markets was carried out by immunoassay, showing an occurrence of boscalid of 15% with a maximum residue concentration of 43 µg/kg.

[ES] Preparación de bioconjugados y anticuerpos para la inmunodetección de anatoxina-a. La presente invención se refiere a bioconjurados y derivados marcados de anatoxina-a, por posiciones diferentes de la molécula, adecuados para la producción de anticuerpos de elevada afinidad y especificidad para anatoxina-a. Asimismo, la presente invención ta,bién se refiere al uso de bioconjurados de anatoxina-a y de derivados marcados de anatoxina-a como antígenos de ensayo. Además, la presente invención también se refiere a métodos de análisis, concentración y extracción de anatoxina-a utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son bioconjurados o derivados de marcados. Esta invención también proporciona un kit para analizar anatoxina-a que comprende anticuerpos frente a esta cianotoxina, en ocasiones junto con antígenos de ensayo que son bioconjugados o derivados marcados de la anatoxina-a., [EN] The present invention relates to bioconjugates and labeled derivatives of anatoxin-a, at different positions of the molecule, suitable for producing antibodies with high affinity and specificity for anatoxin-a. At the same time, the present invention also relates to the use of bioconjugates of anatoxin-a and labeled derivatives of anatoxin-a as assay antigens. Moreover, the present invention also relates to methods for analyzing, concentrating and extracting anatoxin-a using the antibodies obtained, sometimes together with assay antigens which are bioconjugates or labeled derivatives. This invention also provides a kit for analyzing anatoxin-a which comprises antibodies against this cyanotoxin sometimes together with assay antigens which are bioconjugates or labeled derivatives of anatoxin-a.

[EN] The present invention relates to bioconjugates and labeled derivatives of anatoxin-a, at different positions of the molecule, suitable for producing antibodies with high affinity and specificity for anatoxin-a. At the same time, the present invention also relates to the use of bioconjugates of anatoxin-a and labeled derivatives of anatoxin-a as assay antigens. Moreover, the present invention also relates to methods for analyzing, concentrating and extracting anatoxin-a using the antibodies obtained, sometimes together with assay antigens which are bioconjugates or labeled derivatives. This invention also provides a kit for analyzing anatoxin-a which comprises antibodies against this cyanotoxin sometimes together with assay antigens which are bioconjugates or labeled derivatives of anatoxin-a., [ES] Preparación de bioconjugados y anticuerpos para la inmunodetección de anatoxina-a. La presente invención se refiere a bioconjurados y derivados marcados de anatoxina-a, por posiciones diferentes de la molécula, adecuados para la producción de anticuerpos de elevada afinidad y especificidad para anatoxina-a. Asimismo, la presente invención ta,bién se refiere al uso de bioconjurados de anatoxina-a y de derivados marcados de anatoxina-a como antígenos de ensayo. Además, la presente invención también se refiere a métodos de análisis, concentración y extracción de anatoxina-a utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son bioconjurados o derivados de marcados. Esta invención también proporciona un kit para analizar anatoxina-a que comprende anticuerpos frente a esta cianotoxina, en ocasiones junto con antígenos de ensayo que son bioconjugados o derivados marcados de la anatoxina-a.

[EN] The present invention relates to bioconjugates and labeled derivatives of anatoxin-a, at different positions of the molecule, suitable for producing antibodies with high affinity and specificity for anatoxin-a. At the same time, the present invention also relates to the use of bioconjugates of anatoxin-a and labeled derivatives of anatoxin-a as assay antigens. Moreover, the present invention also relates to methods for analyzing, concentrating and extracting anatoxin-a using the antibodies obtained, sometimes together with assay antigens which are bioconjugates or labeled derivatives. This invention also provides a kit for analyzing anatoxin-a which comprises antibodies against this cyanotoxin sometimes together with assay antigens which are bioconjugates or labeled derivatives of anatoxin-a., [ES] Preparación de bioconjugados y anticuerpos para la inmunodetección de anatoxina-a. La presente invención se refiere a bioconjurados y derivados marcados de anatoxina-a, por posiciones diferentes de la molécula, adecuados para la producción de anticuerpos de elevada afinidad y especificidad para anatoxina-a. Asimismo, la presente invención ta,bién se refiere al uso de bioconjurados de anatoxina-a y de derivados marcados de anatoxina-a como antígenos de ensayo. Además, la presente invención también se refiere a métodos de análisis, concentración y extracción de anatoxina-a utilizando los anticuerpos obtenidos, en ocasiones junto con antígenos de ensayo que son bioconjurados o derivados de marcados. Esta invención también proporciona un kit para analizar anatoxina-a que comprende anticuerpos frente a esta cianotoxina, en ocasiones junto con antígenos de ensayo que son bioconjugados o derivados marcados de la anatoxina-a.