Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad.

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.

Commercially available chemicals as immunizing haptens for the development of a polyclonal antibody recognizing carbendazim and other benzimidazole-type fungicides.

Carbendazim is a fungicide widely used for controlling fungi affecting fruits, vegetables, field crops etc. Determination of carbendazim in water, soil and various crops is frequently required to assure compliance with national/European regulations. A polyclonal antibody recognizing carbendazim was developed by using commercially available 2-(2-aminoethyl) benzimidazole, 2-benzimidazole propionic acid and 2-mercaptobenzimidazole as immunizing haptens; each of the above derivatives was directly conjugated to the carrier protein keyhole limpet hemocyanin and a mixture of the conjugates was administered to New Zealand white rabbits. Immunochemical functionality of the antisera and the corresponding isolated antibody (whole IgG fraction) was evaluated through titer and displacement curves in an in-house developed ELISA, which employed a 2-mercaptobenzimidazole - functionalized lysine-dendrimer as the immobilized hapten. As shown with ELISA-displacement curves, the above antibody could recognize carbendazim as well as other benzimidazole-type fungicides, i.e. benomyl and thiabendazole, and also intact benzimidazole, while it did not cross-react with the structurally different pesticides carbaryl and imazalil. Considering the rather simple approach which has led to its development and its highly promising immunochemical profile, the new antibody may be exploited in immunoanalytical systems for detecting benzimidazole-type pesticides e.g. in samples of environmental interest. The above antibody is being currently tested as a biorecognition element in the novel FOODSCAN cell biosensor platform for pesticide residue detection based on the Bioelectric Recognition Assay technology.

Carbon nanotube-protein carriers enhance size-dependent self-adjuvant antibody response to haptens.

Carbon nanotubes (CNTs) are nanomaterials with interesting emerging applications. Their properties make CNTs excellent candidates for use as new nanovehicles in drug delivery, immunization and diagnostics. In the current study, we assessed the immune-response-amplifying properties of CNTs to haptens by using azoxystrobin, the first developed strobilurin fungicide, as a model analyte. An azoxystrobin derivative bearing a carboxylated spacer arm (hapten AZc6) was covalently coupled to bovine serum albumin (BSA), and the resulting BSA-AZc6 conjugate was covalently linked to four functionalized CNTs of different shapes and sizes, varying in diameter and length. These four types of CNT-based constructs were obtained using efficient, fast, and easy functionalization procedures based on microwave-assisted chemistry. New Zealand rabbits and BALB/c mice were immunized with BSA-AZc6 alone and with the four CNT-BSA-AZc6 constructs, both with and without Freund's adjuvant. The IgG-type antibody responses were assessed in terms of the titer and affinity, paying special attention to the relationship between the immune response and the size and shape of the employed CNTs. Immunization with CNT-BSA-AZc6 resulted in enhanced titers and excellent affinities for azoxystrobin. More important, remarkable IgG responses were obtained even in the absence of an adjuvant, thus proving the self-adjuvanting capability of CNTs. Immunogens were able to produce strong anti-azoxystrobin immune responses in rabbits even when administered at a BSA-AZc6 conjugate dose as low as 0.05 µg. The short and thick CNT-BSA-AZc6 construct produced the best antibody response under all tested conditions.

Antibody generation and immunoassay development in diverse formats for pyrimethanil specific and sensitive analysis.

Immunochemical techniques are complementary tools to modern analytical requirements. These methods rely on the production of immunoreagents with adequate binding properties. In the present study, a rationally designed and functionalized derivative of pyrimethanil--a modern anilinopyrimidine fungicide--was synthesized in order to generate for the first time high-affinity and selective antibodies to this xenobiotic. A single coupling procedure--based on hapten activation using N,N'-disuccinimidyl carbonate and purification of the active ester--was followed to prepare both immunizing and assay conjugates. Polyclonal antibodies were produced and characterized by enzyme-linked immunosorbent assay (ELISA) in four alternative formats: one indirect and three direct competitive procedures. The selected immunoassay displayed a limit of detection of 0.024 µg L(-1), far lower than the official maximum residue limits and close to the sensitivity of regular instrumental assays. This ELISA was shown to be robust to buffer changes and tolerant to the presence of little amounts of methanol, ethanol and acetonitrile. Finally, the developed assay was applied to the analysis of pyrimethanil in carrot juice samples, and a limit of quantification of 0.040 mg L(-1) was determined.

Direct surface plasmon resonance immunosensing of pyraclostrobin residues in untreated fruit juices.

A surface plasmon resonance (SPR) immunoassay for on-line detection of the strobilurin fungicide pyraclostrobin in untreated fruit juices is presented. The analysis of pyraclostrobin residues is accomplished in apple, grape, and cranberry samples by monitoring the recognition events occurring separately in a two-channel home-made SPR biosensor. Covalent coupling of the analyte derivative results in a reversible method, enabling more than 80 measurements on the same sensor surface. Optimization of the immunoassay conditions provides limits of detection as low as 0.16 µg L(-1). The selectivity and reproducibility of the analysis is ensured by studying both non-specific interactions with unrelated compounds and inter-assay coefficients of variation. Excellent recovery ranging from 98 to 103% was achieved by a simple 1:5 dilution of fruit juice with assay buffer before the analysis. The lack of previous cleaning and homogenization procedures reduces the analysis time of a single food sample to only 25 min, including the regeneration cycle.

Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays.

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).

Synthesis of azoxystrobin transformation products and selection of monoclonal antibodies for immunoassay development.

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.

Development of competitive immunoassays to hydroxyl containing fungicide metabolites.

This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 µg/L and a limit of detection (LOD) of 1.1 µg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 µg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 µg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.

Hapten synthesis, monoclonal antibody generation, and development of competitive immunoassays for the analysis of picoxystrobin in beer.

This paper describes the original synthesis of a functionalized derivative of the fungicide picoxystrobin and the generation of the first reported monoclonal antibodies against this strobilurin pesticide. The synthetic hapten was prepared by total synthesis from commercial chemicals and incorporating the spacer arm through a carbon-carbon single bond. Also, to obtain the immunogen, an uncommon hapten activation strategy based on N,N'-disuccinimidyl carbonate was employed, affording high activation yields and clean and reproducible coupling results. With these immunoreagents, two enzyme-linked immunosorbent assays (ELISAs) were developed: a competitive one-step assay using the antibody-coated direct ELISA format and a competitive two-step assay with the conjugate-coated indirect ELISA procedure. Both immunoassays were characterized in terms of sensitivity, selectivity, tolerance to solvents and matrix effects, achieving limits of detection below 0.2 µgL(-1). The optimized assays were used for the determination of picoxystrobin residues in beer, with recovery values ranging between 90 and 121% for the direct assay and from 79 to 122% for the indirect assay.

Contact allergy to thiurams: multifactorial analysis of clinical surveillance data collected by the IVDK network.

PURPOSE: To analyse the association between occupation (represented by job title) and contact allergy to thiuram vulcanising agents based on data of a clinical registry (IVDK, www.ivdk.org ). METHODS: Clinical, demographic and allergy patch test data of all patients tested between 1992 and 2006 with the thiuram mix (1% in petrolatum) as part of the baseline series was analysed (n = 121,051). Poisson regression analysis was used to quantify the association between different occupations (and other relevant factors) and a positive patch test reaction to the thiuram mix. Furthermore, the time trend of sensitisation prevalence was analysed in high-risk occupational subgroups identified. RESULTS: In comparison to a largely unexposed reference group (office workers and teachers), rubber manufacturers had a significantly elevated risk (prevalence ratio (PR): 5.1, 95% confidence interval (CI) 2.0-10.5). However, health care workers such as physicians and dentists (PR: 3.8, 95% CI: 3.0-4.8) or nursing staff (PR: 3.0, 95% CI: 2.5-3.6) as well as meat and fish processors (PR 3.5, 95% CI: 2.2-5.3) and cleaners (PR 3.1, 95% CI: 2.5-3.8) were found to have a high sensitisation risk as well. In case of health care workers, a significant downward trend during the study period was observed; while in food processors and cleaners, sensitisation prevalence remained largely stable. CONCLUSION: The adjusted multifactorial analysis identified occupations yet unknown to be associated with elevated thiuram contact allergy risk, e.g., food processors and cleaners. Thus, (i) further in-depth research can be targeted and (ii) efforts to prevent sensitisation to thiurams focussed, e.g., by limiting thiuram concentrations in products to a residual level which is technically inevitable.

Development of monoclonal immunoassays for the determination of triazole fungicides in fruit juices.

Enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for the detection of triazole fungicides have been developed. With this aim, hapten-protein conjugates, containing the common triazole and chlorinated aromatic moieties, were prepared. From mice immunized with these conjugates, several monoclonal antibodies (MAbs) with the ability to sensitively bind several triazoles with different specificity were obtained. Both analyte- and class-specific ELISAs were developed. The hexaconazole-specific immunoassay can determine this fungicide with a limit of detection of 0.3 mug/L in standard buffer. The so-called triazole-specific immunoassay allowed for the detection of tetraconazole, penconazole, cyproconazole, and myclobutanil, with limits of detection in the 0.1-0.7 mug/L range. These immunoassays were applied to the determination of triazoles in spiked fruit juices. Samples were adequately diluted to minimize the matrix effects. Coefficients of variation were below 30%, and recoveries ranged from 62 to 135%. Therefore, the developed immunoassays can determine triazole fungicides in fruit juices down to the maximum residue limits currently legislated, without any sample treatment other than dilution.

Production and characterization of monoclonal antibodies specific to the strobilurin pesticide pyraclostrobin.

Strobilurin fungicides are nowadays among the most important fungicides in the market of active agrochemicals. Pyraclostrobin, which belongs to the last generation of this family of molecules, shows a broader antifungal activity spectrum and higher efficiency and security profiles than previous fungicides. This paper describes the synthesis of functionalized haptens, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays (ELISA) for the detection of pyraclostrobin. A conformational analysis of hapten structure was performed, which provided relevant data concerning the length of the spacer arm. A very useful strategy has been followed for the screening of hybridomas, leading to the selection of a panel of high-affinity monoclonal antibodies to pyraclostrobin. Moreover, different immunoassays have been characterized using the conjugate-coated indirect ELISA format, and limits of detection below 0.1 microg/L have been obtained. Also, a simplified one-step procedure has been carried out with two indirect assays. Finally, these results have been compared with the performance of the same antibodies in the antibody-coated direct ELISA format.

Differential detection of N-heterocyclic compounds and their N-methylated derivatives by immunoanalysis.

Competitive enzyme-linked immunosorbent assay (ELISA) systems are proposed for the indirect monitoring of formaldehyde by the parallel detection of its N-methylated precursors and the corresponding demethylated compounds. As an example for such immunoanalytical differentiation between an N-heterocyclic compound and its N-methylated derivative, the quantitative detection of the systemic triazole fungicide, myclobutanil, is discussed. Antibodies recognizing the non-zwitterionic structure of 2-(4-chlorophenyl)-2-[(1,2,4-triazol-1-yl)-methyl]-hexanonitril e (myclobutanil) showed only minor binding to corresponding N-alkylated derivatives of myclobutanil. And vice versa, literature data indicate that antibodies raised against the pyridilium ionic structure of the herbicide paraquat, displayed only mediocre reactivity towards the corresponding dealkylated derivatives. Thus, both experimental and literature data suggest that immunoanalytical methods for differential detection of N-methylated heterocycles (potentially including formaldehyde precursors) and their non-methylated counterparts are possible to develop.

Immunomodulatory effects of occupational exposure to mancozeb.

The effects of occupational exposure to ethylene-bis-dithiocarbamate of manganese and zinc on the immune system were evaluated in a group of mancozeb-exposed manufacturers and controls. The immune system tests revealed the following: (a) lymphocyte proliferative responses triggered by different activators and mitogen-induced IL-2 production were higher in exposed subjects than in controls; (b) production of monocyte/macrophage-derived IL-1 and polyclonal IgG and IgM, by beta-lymphocytes, did not differ between exposed subjects and controls; (c) percentages and absolute numbers of total T-cells, T-helper cells, T-suppressor/cytotoxic cells, activated T-cells, total beta-cells, and natural killer cells were similar in exposed subjects and controls; (d) serum immunoglobulin classes and complement fractions were within the range of normality; and (e) rheumatoid factor and non-organ-specific serum autoantibodies were absent in exposed and control subjects. An increase in T-cell functional response was found in the exposed group, suggesting a slight immunomodulator effect of mancozeb in conditions of low-level, prolonged occupational exposure.

Post harvest control of aflatoxin production on in-shell moist peanuts by sodium ortho-phenylphenate. II. Harvester tests

O objetivo deste trabalho foi o de otimizar o sistema de pulverizaçäo, em campo, da soluçäo de ortofenilfenato de sódio (OFS) sobre amendoim em casca, para verificar a eficiência desta substância no controle da produçäo de aflatoxinas. Em trabalho realizado anteriormente por Fonseca et al. (6) verificou-se que a pulverizaçäo sob condiçöes de campo foi deficiente uma vez que a cobertura completa da vagem, com a soluçäo de OFS näo foi conseguida, indicando assim a necessidade de otimizaçäo desta operaçäo. Deste modo, na safra das águas de 1988, a pulverizaçäo foi realizada na própria colhedora mecânica onde o sistema de ulverizaçäo foi adaptado. A concentraçäo da soluçäo de OFS utilizada foi de 0,5 por cento. Nesta safra, a despeito da melhor cobertura das vagens com a soluçäo ocorreu o acúmulo de vagens na bica de saída da colhedora, por ocasiäo da troca da sacaria já cheia pela vazia, o que prejudicou a pulverizaçäo, dificultando a cobertura de todas as vagens com a soluçäo. Observou-se que o teor inicial de aflatoxinas aumentou durante o período de armazenamento, tanto nos lotes tratados como nos lotes de controle. A alta contaminaçäo com aflatoxinas pode ter ocorrido por näo se ter obtido ainda uma pulverizaçäo perfeita de todas as vagens e/ou devido à concentraçäo insuficiente da soluçäo de OFS para o controle da produçäo da toxina

A test series for pesticide dermatitis.

A pesticide patch test series was tested in 200 subjects, 50 of whom were agricultural workers. Positive reactions to fungicides were found in 24, almost all to thiophthalimides, especially captan, difolatan and folpet. Reactions to bis-dithiocarbamates and benomyl were rare, and to other pesticides not significant.