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Fungal plant pathogens have posed a significant threat to crop production. However, the large-scale application of pesticides is associated with possible risks for human health and the environment. Boscalid is a widely used fungicide, consistently implemented for the management of significant plant pathogens. Conventionally, the detection and determination of boscalid residues is based on chromatographic separations. In the present study, a Bioelectric Recognition Assay (BERA)-based experimental approach combined with MIME technology was used, where changes in the electric properties of the membrane-engineering cells with anti-boscalid antibodies were recorded in response to the presence of boscalid at different concentrations based on the maximum residue level (MRL) for lettuce. The membrane-engineering Vero cells with 0.5 μg/mL of antibody in their surface were selected as the best cell line in combination with the lowest antibody concentration. Furthermore, the biosensor was tested against another fungicide in order to prove its selectivity. Finally, the BERA cell-based biosensor was able to detect the boscalid residue, below and above the MRL, in spiked lettuce leaf extracts in an entirely distinct and reproducible manner. This study indicates that the BERA-based biosensor, after further development and optimization, could be used for the routine, high-throughput detection of boscalid residue in lettuce, and not only that. [ABSTRACT FROM AUTHOR]

Appropriate hapten design and synthesis have been identified as critical steps to generate 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Early warning systems for monitoring toxic events may benefit from the availability of monoclonal antibodies enabling the sensitive and specific detection of anatoxin‐a, a cyanotoxin involved in numerous cases of animal poisoning resulting from toxic algal blooms in freshwaters. Through the synthesis of three functionalized derivatives of anatoxin‐a, we have succeeded in generating the first‐ever reported immunoreagents (bioconjugates and antibodies) suitable for the development of immunoanalytical approaches aimed at rapid and onsite detection of this harmful cyanotoxin. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) is a carcinogenic mycotoxin produced by Aspergillus and Penicillium, which contaminates cereals like corn and wheat, in addition to beverages such as coffee, wine and beer. Chromatography is the preferred analytical method for OTA determination, but unfortunately this method is still elaborate, expensive and tedious. Alternative mycotoxin detection techniques involve assays and sensors based on molecularly imprinted polymers nanoparticles (nanoMIPs) as synthetic polymeric receptors mimicking antibodies. Herein we designed nanoMIPs for specific recognition of OTA using computational screening and docking of different acryl functional monomers. Subsequently, different nanoMIPs were prepared by solid phase synthesis using two OTA haptens, OTAe‐MIPs without carboxylic group and OTAb‐MIPs bearing the carboxylic group. NanoMIPs affinity towards OTA was analyzed by surface plasmon resonance, the results displayed dissociation constants for OTAb‐MIPs at 0.4 μM and for OTAe‐MIPs at 5.1 μM. Therefore, the results demonstrate that nanoMIPs recognition depends on the immobilization position of the OTA hapten as observed using antibodies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the original publication [[1]], Scheme 1 and Scheme 2 are misplaced due to mistakes in the publication process. Graph: Scheme 2 Synthesis of AL b -NHS ester. The correct schemes should be as follows: Graph: Scheme 1 Synthesis of AL a -NHS ester. [Extracted from the article]

Bioconjugate 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Boscalid is a modern agrochemical belonging to the so-called chemical class of succinate dehydrogenase inhibitor fungicides. With the aim of developing rapid analytical screening methods for this relevant compound, we herein report the synthesis of new boscalid mimics and the study of their suitability for the production of polyclonal antibodies. Aliphatic spacer arms equivalent in length and composition were tethered at two different aromatic rings of the target molecular structure. These haptens, besides being used for immunization, were employed in the development of heterologous competitive enzyme-linked immunosorbent assays (cELISAs) in order to improve assay detectability. Direct and indirect immunoassays were tailored and applied to the determination of samples with incurred boscalid residues. The assays were characterized in terms of sensitivity, specificity, trueness, and precision. Limit of quantification was established at 5 μg kg−1, coefficients of variation were lower than 20%, and recoveries from spiked samples ranged from 90 to 137%. Finally, ELISA performance was evaluated by Deming regression analysis with tomato and cucumber samples, selecting ultra-performance liquid chromatography-mass spectrometry as the reference method. The results showed that the proposed cELISAs are useful for the routine determination of boscalid fungicides in foods with high-sample throughput and affordable cost. [ABSTRACT FROM AUTHOR]