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.
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.
A solid state formulation of Beauveria bassiana (Balsamo) Vuillemin has been developed for biological control of the Red Palm Weevil (RPW), Rhynchophorus ferrugineus (Olivier, 1790). Two kinds of ...bioassays (dry conidia and dipping) using 10 isolates from several coleopterans in Mediterranean environments, identified 2 RPW derived isolates (193 and 203) as most pathogenic to RPW larvae and adults (zero survival within first 4–5 d for dry conidia, and 14 and 23 d for dipping bioassays). Isolate 203 (5.1 × 108 ± 1.9 × 108 conidia g-1) was formulated with fragmented date seed into solid granules and tested in palms infested with RPW under semi-field conditions in Feb, Apr/May and Jun of both 2007 and 2008. Beauveria bassiana significantly reduced RPW adult survival with respect to controls in May 2007 and in the Apr/Jun 2008 experiments. Total RPW adult mortality was achieved within 30 days for all B. bassiana treatments, and was associated with increasing numbers of insects with signs of mycosis in 2008 experiments. Beauveria bassiana formulation reduced RPW multiplication in artificially infested palms compared to controls, and a positive correlation between numbers of larvae and time post-infestation was recorded. The suppression of RPW adult populations by B. bassiana persisted for at least 3 months under semi-field conditions. The Beauveria bassiana solid formulation, which induces great adult mortality and persistence in the field, could be applied as a preventive as well as a curative treatment for the integrated management of RPW.
Old Silks Endowed with New Properties Plaza, Gustavo R; Corsini, Paola; Marsano, Enrico ...
Macromolecules,
11/2009, Letnik:
42, Številka:
22
Journal Article
Recenzirano
High-performance regenerated silkworm Bombyx mori silk fibers with new properties that mimic those of spider silk can be produced through a wet spinning process modified with an immersion ...postspinning drawing (IPSD) step. IPSD fibers show the ability to recover from irreversible deformation, and their tensile behavior can be tailored repeatedly, features solely exhibited until now by natural spider silk. It is further shown that the new properties emerge from a microstructure that is closer to that of spider than to natural silkworm silk. This work demonstrates that processing plays a role at least comparable to that of the amino acid sequence in the final properties of the material. The spinning process does not only modify the mechanical parameters of the fiber but also can even prompt the emergence of new properties, opening a wide range of new applications for regenerated silk fibers. It also represents a significant change of the paradigm in the field of biomimetics, given that it relaxes the condition of copying the natural protein sequences as close as possible to recover the outstanding properties of the natural materials.
► 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.
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.
Anatoxin-a and homoanatoxin-a are highly neurotoxic compounds produced by cyanobacteria, principally during surface water-blooms (SWBs). Owing to their powerful biological activity and unique ...structural characteristics, these natural alkaloids have been the subject of extensive research work in both pharmacological and synthetic studies. In this contribution we report a simple and efficient synthetic approach for the preparation of both the natural and unnatural enantiomers of these cyanotoxins, (+)-1 and (+)-2 and (−)-1 and (−)-2 respectively. Key features of this approach include: i) construction of the azabicyclic homotropane framework in both enantiomeric forms from cis-5,6-epoxycyclooctene, based on a microwave mediated epoxide ring opening reaction by a chiral benzyl amine followed by a transannular amine-alkene cyclization; ii) elaboration of the characteristic methyl or ethyl enone by means of a Sonogashira cross-coupling reaction of an enol-triflate with a C2 or C3 terminal alkyne, followed by a chemo- and regio-selective hydration of the resulting conjugated enyne group.
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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.
Purpose
This study aims to asses a cancer fast-track programme (CFP) to shorten the time since a patient with suspicion of cancer is referred by the primary care (PC) physician to the specialized ...medical team.
Methods
Guidelines for main suspected tumours were designed to help PC physicians to detect and rapidly refer cases to the CFP oncology coordinator, who sent them to the appropriate department to accelerate diagnosis, staging and therapy. All patients analysed in this report were referred from June 2009 to July 2012.
Results
A total of 897 suspected cancer cases were submitted and finally 705 were studied. In 205 (29 %) a cancer diagnosis was confirmed within 23 days (median). Therapy was initiated within 46 days after referral (median). Early diagnoses with a potential curative approach were made in 166 (82 %).
Conclusions
This CFP decreased the waiting time for cancer diagnosis, by improving communication between PC physician and specialized care teams. Most patients included in this program could get therapy with curative intent.
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