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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.