Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to ...strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin.
Fluxapyroxad is a new-generation carboxamide fungicide, with residues increasingly being found in food samples. Immunochemical assays have gained acceptance in food quality control as rapid, ...cost-effective, sensitive, and selective methods for large sample throughput and in situ applications. In the present study, immunoreagents to fluxapyroxad were obtained for the first time, and competitive immunoassays were developed for the sensitive and specific determination of fluxapyroxad residues in food samples. Two carboxyl-functionalized analogues of fluxapyroxad were prepared, and antibodies with IC50 values in the low nanomolar range were generated from both haptens, though a dissimilar response was observed concerning specificity. A robust direct assay was set up, with a calibration curve exhibiting a limit of detection of 0.05 nM (0.02 μg/L). Limits of quantitation of 5 μg/L were obtained for peach, apple, and grape juices using samples diluted in water. The direct immunoassay was also successfully applied to the determination of fluxapyroxad in grapes from in-field treated grapevines.
Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that ...both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC
value for the sum of spirotetramat and spirotetramat-enol of 0.1 μg/L was developed using selected antibodies and a novel heterologous bioconjugate carrying a rationally-designed hapten. Studies with fortified grape, grape juice, and wine samples showed good precision and accuracy values, with limits of quantification well below the maximum residue limits. Excellent correlation of results was observed with a standard reference chromatographic method. As a step forward, a lateral flow immunoassay was developed for onsite screening analysis of spirotetramat in wine. This assay was successfully validated according to Regulation 519/2014/EU for semi-quantitative methods at concentrations in line with the legal levels of spirotetramat and spirotetramat-enol in grapes, with a satisfactory false suspect rate below 2%.
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.
Bioorthogonal reactions have revolutionized the way low-molecular-weight compounds are coupled to biomolecules. Organic chemistry, polymer science, and chemical biology are among the disciplines that ...have benefited the most from this breakthrough. Despite the reliability of the click chemistry concept for the efficient and chemoselective functionalization of biomacromolecules with haptens at preferred positions, the fact that azide–alkyne cycloaddition reactions originate new chemical moieties as part of the linker may have delayed their application in the immunodiagnostic field. Using the mycotoxin ochratoxin A as a model compound, we herein demonstrate for the first time that bioconjugates arising from the ligation between an azido-bearing hapten and an alkyne-modified carrier protein are able to elicit the generation of high-affinity monoclonal antibodies suitable for the development of rapid methods for the immunodetection of small organic molecules.
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.
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•A novel hapten with the linker at C-1 was designed for the mycotoxin alternariol.•Specific, high-affinity monoclonal antibodies to alternariol were generated.•A heterologous immunoassay was optimized for alternariol analysis.•The LOQ was 1 ng/mL for oats and buckwheat flour and 10 ng/mL for pears.•The immunoassay was statistically validated using pears infected with Alternaria.
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.
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•Dicamba has emerged as a relevant contaminant due to weed resistance to glyphosate.•A collection of haptens with different linkers and tethering sites was designed.•Monoclonal antibodies specific of dicamba were generated for the first time.•A highly sensitive direct competitive ELISA was developed for dicamba analysis.•Dicamba residues were successfully determined in environmental water samples.
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.
•ELISA and LFIA based on novel immunoreagents were developed for OTA detection in wine and must.•The ELISA allows the analysis of OTA at concentrations below the limit set by the EU (2 ng/mL).•The ...LFIA showed 80% and complete inhibition at 0.5 and 2 ng/mL, respectively.•LFIA was validated following EU Regulation 519/2014 for semi-quantitative screening methods.•The immunoassays are suitable for on-site monitoring of samples contaminated with OTA.
Immunochemical methods are highly deployed in analytical laboratories worldwide for monitoring the incidence of mycotoxins in the food chain. Nevertheless, most conventional immunoassays for ochratoxin A (OTA), including commercial kits, show limitations to robustly determine this mycotoxin in grape-derived products below regulated levels (2 ng/mL). Herein, two rapid tests for sensitive OTA determination in wine and must were developed capitalizing on a collection of bioconjugates from innovative synthetic haptens and monoclonal antibodies with subnanomolar affinity. The ELISA (LOD = 8 pg/mL) showed excellent performance in recovery studies, and it was applied to survey commercial wines and musts for OTA contamination. Concerning LFIA, validation according to the Commission Regulation 519/2014 showed that samples exceeding 2 ng/mL were properly scored as uncompliant. More importantly, illegal samples provided a complete inhibition of the test signal, making this test an easy-to-use, rapid, and convenient screening method for in-house control of OTA in wineries.
Carbon nanotubes are novel technological tools with multiple applications. The interaction between such nanoparticles and living organisms is nowadays a matter of keen research by academic and ...private institutions. In this study, carbon nanotube constructs were investigated as delivery vehicles for immunostimulation and induction of the secondary immune response to a small organic molecule, namely, a hapten. Two types of nanoconstructs were prepared: on one hand, carbon nanotubes carrying a protein bioconjugate of a hapten covalently linked to the carbon surface, and on the other hand, covalent carbon nanotube constructs of the same model chemical compound without the carrier protein. Nanotube vehicles carrying a hapten–protein bioconjugate were demonstrated to stimulate the immune system and to induce a strong primary immune response against the hapten with as low as 0.1 μg of the model chemical. The influence of the different elements of those nanoconstructs over the immune response was investigated to better understand the molecular mechanisms that are involved. As expected, the presence of the carrier protein was shown to be necessary in order to trigger the immune response. Interestingly, we found that a remarkable secondary immune response to the model organic compound occurred in the absence of a carrier protein. Additionally, a satisfactory adjuvant effect of carbon nanotubes was observed and a potent immune response was elicited without employing an oil-based adjuvant.
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