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.
Spirotetramata tetramic acid insecticideis 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.
Kresoxim-methyl is nowadays widely used to combat a diversity of common diseases affecting high-value crops. In this article, we report the development and characterization of two novel immunoassays ...for the analysis of this pioneer strobilurin fungicide, and for the first time, a validation study with food samples was performed. A direct and an indirect competitive immunoassay based on a new anti-kresoxim-methyl monoclonal antibody were developed for sensitive and specific chemical analysis. Optimized assays showed limits of detection of 0.1 μg/L. Fruit and vegetable samples were extracted with acetonitrile by the QuEChERS procedure and analyzed by the developed immunoassays after a simple dilution in buffer, affording limits of quantification below US and European maximum residue limits. Immunochemical results of samples from kresoxim-methyl-sprayed strawberry fields demonstrated good statistical agreement with gas chromatography coupled to mass spectrometry as reference technique.
Pyraclostrobin belongs to a new generation of fungicides widely used to preserve high valuable crops. In the present study, three monoclonal antibodies with different affinities to this modern ...strobilurin have been evaluated for their usefulness in the production of immunoaffinity columns suitable for the solid-phase extraction, concentration, and clean-up of residues from food commodities. Different immunosorbents were produced and characterized in terms of antibody immobilization efficiency, immunosorbent binding capacity, optimum elution conditions, and reusability. Covalent coupling of the antibodies to Sepharose–CNBr gel took place with high yield (over 90%), whereas the immunosorbent efficacy to retain the analyte (from 28 to 68%) was shown to depend on the amount and type of antibody immobilized on the support. As a matter of fact, columns prepared with the monoclonal antibody PYs5#14 were able to selectively bound up to 53
μg of pyraclostrobin per gram of beads. Acetonitrile solutions were preferred over methanolic ones for analyte elution, and some immunosorbents could be reused at least 4–6 times provided that the amount of pyraclostrobin and the volume of sample did not overload the column. Effectiveness of the selected immunoaffinity column was evidenced by the development of an extraction procedure for pyraclostrobin residues from fruit juices and further determination by high-performance liquid chromatography with UV detection. A concentration factor of 50 times was achieved with the developed immunoaffinity column, which eventually resulted in a limit of quantification of 0.01
mg
L
−1. Finally, quantitative recoveries were obtained on apple juice and red grape must samples spiked with pyraclostrobin from 0.01 to 1
mg
L
−1.
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 0.3 μg/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.
•Pyrimethanil is an anilinopyrimidine fungicide very often found in food.•We herein described the first-ever reported monoclonal antibodies to pyrimethanil.•Two novel regioisomeric pyrimethanil mimics were designed and synthesized.•Competitive immunoassays in different formats were developed.•ELISA method was validated by comparing analytical results with UPLC–MS–MS.
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
), 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.
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.
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► Four azoxystrobin regioisomeric haptens were used to immunize mice. ► A collection of specific monoclonal antibodies was generated. ► The developed competitive immunoassay had a LOD ...of 17
ng
L
−1 for azoxystrobin standards. ► The optimized assay was validated with fortified and real food samples.
Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and heterologous antigens, and the immunoassay consisting of monoclonal antibody AZo6#49 and the coating conjugate OVA–AZb6, which displayed an IC
50 value of 0.102
μg
L
−1 and a LOD of 0.017
μg
L
−1, was eventually optimized. The response to different pH and ionic strength conditions of the specific assay was studied using a biparametric approach. In addition, the influence of Tween 20 and organic solvents over the assay parameters was also evaluated. After optimization, the developed immunochemical assay was applied to the analysis of azoxystrobin in spiked juices of relevant fruits and vegetables, showing excellent recoveries between 2 and 500
μg
L
−1.
► Five of the most relevant transformation products of azoxystrobin were synthesized and thoroughly characterized. ► The synthesized compounds were employed as ligands for screening a collection of ...anti-azoxystrobin monoclonal antibodies. ► An antibody (mAb AZa6#210) was identified displaying subnanomolar affinity to three azoxystrobin breakdown products. ► The selected binder is the first ever reported antibody against azoxystrobin transformation products. ► The described generic monoclonal antibody is suitable for implementation in different immunoanalytical platforms.
The use of agrochemicals for crop protection may result in the presence of toxic residues in soils and aquatic environments, besides in foodstuffs. Most often just the parent compound is included in the definition of pesticide residue, even though chemicals resulting from biotransformation and degradation routes might also be of toxicological relevance. Azoxystrobin is a broad-spectrum systemic fungicide widely used worldwide to combat pathogenic fungi affecting plants. We herein report the synthesis and detailed chemical characterization of several of the most relevant metabolites and degradates of azoxystrobin. These compounds were further employed as ligands for screening a collection of monoclonal antibodies to azoxystrobin, which had been previously generated from haptens functionalized at different positions of the target chemical. As a result, an antibody was identified capable of binding, with subnanomolar affinity, not only azoxystrobin but also its main transformation products, such as the so-called acid and enol derivatives, as well as the azoxystrobin (Z)-isomer. The selected binder was demonstrated as a useful immunoreagent for the development of immunochemical assays as novel analytical tools for the qualitative determination of azoxystrobin and its metabolites and degradates.
Cyprodinil is a new-generation anilinopyrimidine fungicide widely used in crop protection and frequently found in fruits. In this study, novel derivatives of cyprodinil with linker site heterologies ...were synthesized and employed in order to produce antibodies with enhanced affinity. Moreover, moiety-heterologous haptens were designed and prepared for assay sensitivity improvement. Two competitive enzyme-linked immunosorbent assays for the analysis of this active substance were developed using direct and indirect formats, achieving IC50 values around 0.15 μg/L. Analytical figures of merit and usability of the optimized assays were evaluated with wine and cider as model food processed matrices. The obtained recoveries were from 90% to 120%, and the limit of quantification was in the 1–5 μg/L range. Finally, a monitoring study (n = 150) was performed to estimate the occurrence and the concentration of cyprodinil in commercial wine and cider products from different origins. We found that 28% of the analysed wine samples contained cyprodinil residues at levels higher than 5 μg/L.
•Regioisomeric haptens with alternative linker sites were prepared for immunization.•Moiety-heterologous haptens were synthesized for immunoassay sensitivity enhancement.•High-sensitivity assays for cyprodinil were developed, with IC50 values below 1 μg/L.•Direct and indirect cELISAs were optimized for trace analysis in wines and cider.•Residues were encountered in 28% of commercial wine samples from different origins.
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