Abstract
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.
Highlights
Antibody-based detection techniques are currently invaluable analytical tools in numerous disciplines, including basic biochemical and biomedical research, forensic toxicology, clinical diagnostics, food safety, and environmental monitoring
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
Hapten synthesis is usually deemed the key step in the generation of antibodies to small organic chemicals, so easy access to suitable functionalized derivatives of target compounds greatly facilitates the production of anti-hapten antibodies and the development of immunochemical methods
Summary
Antibody-based detection techniques are currently invaluable analytical tools in numerous disciplines, including basic biochemical and biomedical research, forensic toxicology, clinical diagnostics, food safety, and environmental monitoring. In the last few years, we have reported the application of the Sonogashira cross-coupling reaction to the successful preparation of conveniently functionalized derivatives of various modern agrochemicals [3,4,5,6,7,8,9,10,11] In all these cases and as a key step in each particular synthetic strategy, previous preparation of a halogenated intermediate was required because all of the concerned analytes were lacking from the proper halogen moiety for further introduction of the corresponding spacer arm via cross-coupling chemistry. Performance and increased stability to sunlight, allowing field application at low rates [16] This fact, together with its novelty and efficacy as fungicide, makes proquinazid an outstanding candidate to study the adequacy of cross-coupling reactions to the synthesis of adequate functionalized mimics that enable to trigger specific and strong immune responses and eventually afford high-affinity and specific antibodies. Performance of the resulting antisera in terms of affinity, specificity, and bioanalytical capability was evaluated by competitive enzyme-linked immunosorbent assay (cELISA)
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