One-step icELISA developed with novel antibody for rapid and specific detection of diclazuril residue in animal-origin foods.

Diclazuril, a broad-spectrum anticoccidial drug, may be accumulated in edible tissues of animals through illegal use, which poses potential threats to human health through the food chain. In this study, an innovative hapten was designed and an immunogen of diclazuril was successfully synthesised with keyhole limpet haemocyanin as carrier protein; then a monoclonal antibody with high specificity was obtained. Furthermore, based on the novel antibody, a one-step indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for rapid and specific detection of diclazuril residues. Compared with the traditional icELISA method, this method saves at least 0.5 hours and one washing step. Under the optimal conditions, the one-step icELISA for diclazuril exhibited good performance with a 50% inhibition concentration (IC50) value of 0.952 µg/kg. The average recoveries of the icELISA ranged from 73.1% to 115.5% with the coefficient of variation lower than 12.7%, which was evaluated by detecting spiked animal-origin food samples. Finally, the one-step icELISA shows a good correlation with an ultra-high liquid chromatography-tandem mass spectrometry method. Those results demonstrate that the one-step icELISA developed for diclazuril detection is time-saving, low-cost, specific, sensitive, and reliable. It shows good potential for social, environmental, and economic benefits in future use.

Quick and convenient construction of lambda-cyhalothrin antigen for the generation of specific antibody.

Lambda-cyhalothrin is a pyrethroid widely used in crop, fruit and vegetable production, but has potential health threats to human. Immunoassay is a cheap, rapid and facile method to detect lambda-cyhalothrin, yet wide application of this method still requires improvement in the construction of antigen. In this study, we developed a one-step lambda-cyhalothrin hapten synthesis that transformed the cyanide group in lambda-cyhalothrin to amide. Complete antigen was assembled by coupling the amide with succinic-anhydride-activated carrier proteins, and corresponding polyclonal antibodies were generated using Balb/c mice. Using antibody generated by the method in this paper, the competitive ELISA demonstrated the lowest detection limit of 3.772 µg/L for lambda-cyhalothrin, and no significant cross-reactivity for other pyrethroid pesticides was observed. All the results suggested we have established a more efficient technique of generating lambda-cyhalothrin antibody. Furthermore, since the activated proteins used in this study are highly controllable, we believe these proteins could potentially be the prototype of a series of standardized carrier proteins for the synthesis of complete antigens.

Occupational contact sensitization in female geriatric nurses: Data of the Information Network of Departments of Dermatology (IVDK) 2005-2014.

BACKGROUND: Geriatric nurses (GN) have a high risk of occupational contact dermatitis (OCD), with chronic irritant contact dermatitis predominating. However, allergic contact dermatitis is an important issue as well. Little is known whether the relevant occupational allergen spectrum reported in the 1990s, including fragrances, preservatives, rubber chemicals and ingredients of surface disinfectants to be the most common sensitizers in GN, is still valid. OBJECTIVES: To monitor the current allergen spectrum in GN with OCD and verify the validity of the patch test recommendations (baseline-, preservative-, ointment base-, rubber-, disinfectant, series and fragrances) in GN with suspected OCD given by the German Contact Dermatitis Research Group (DKG). METHODS: Retrospective analysis of IVDK data (2005-2014) of 743 female GN with OCD, in comparison to 695 GN without OCD. RESULTS: GN with OCD reacted significantly more frequently to both fragrance mixes, hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC), thiuram mix, zinc diethyldithiocarbamate and mercaptobenzothiazole than GN without OCD. Reactions to MDBGN, methylchloroisothiazolinone/methylisothiazolinone and oil of turpentine occurred substantially, but not significantly more frequently among GN with OCD. The latter may be due to former use of a special alcoholic liniment in geriatric care. Among material from the patients' workplaces, tetrazepam was a frequent allergen, due to dust exposure from pill crushing. Furthermore, occupationally used protective gloves, body care products as well as surface disinfectants were often tested positively. CONCLUSIONS: The general allergen spectrum in GN with OCD is unchanged, so the DKG patch test recommendations are still valid. Prevention of occupational sensitization should focus on fragrance-free hygiene and body care products, usage of accelerator-free protective gloves and avoidance of drug dust exposure.

Development of an Immunosensor for Determination of the Fungicide Chlorothalonil in Vegetables, Using Surface Plasmon Resonance.

An immunosensor based on surface plasmon resonance (SPR-sensor) was developed to analyze chlorothalonil residues and maximum residue limits (MRLs; 0.5-50 mg/kg) in vegetables in Japan. Conjugates of N-(pentachlorophenoxyacetyl)glycine and bovine serum albumin were covalently coated on the sensor chip. The SPR-sensor quantitatively determined chlorothalonil at concentrations ranging from 8.0 to 44 ng/mL, using TPN9A, a monoclonal antibody to chlorothalonil. The 50% inhibition concentration was 25 ng/mL. The reactivity was 10-fold lower than that of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). However, the SPR-sensor could determine chlorothalonil residues in vegetables at concentrations around the above MRLs. Chlorothalonil spiked in vegetables was recovered at 90-118% within 1 day and at 90-115% across 3 days, correlating with HPLC results. The sensor showed good performance for chlorothalonil residue analysis in vegetables with rapid determination, although the sensitivity and the cross-reactivity were less effective than with the ic-ELISA.

Synthesis and Characterization of Hapten-Protein Conjugates for Antibody Production against Cyanogenic Glycosides.

Consumption of cyanogenic plants can cause serious health problems for humans. The ability to detect and quantify cyanogenic glycosides, capable of generating cyanide, could contribute to prevention of cyanide poisoning from the consumption of improperly processed cyanogenic plants. Hapten-protein conjugates were synthesized with amygdalin and linamarin by using a novel approach. Polyclonal antibodies were generated by immunizing four New Zealand White rabbits with synthesized amygdalin-bovine serum albumin and linamarin-bovine serum albumin immunogen. This is the first time an antibody was produced against linamarin. Antibody titer curves were obtained from all the four rabbits by using a noncompetitive enzyme-linked immunosorbent assay. High antibody titer was obtained at dilutions greater than 1:50,000 from both immunogens. This new method is an important step forward in preventing ingestion of toxic cyanogenic glycosides.

Development of direct competitive ELISA for residue analysis of fungicide chlorothalonil in vegetables.

A direct competitive (dc)-ELISA was developed for rapid and simple determination of chlorothalonil residue in vegetables. A carboxylic acid derivative of pentachlorophenol was used to prepare an anti-chlorothalonil monoclonal antibody (MoAb) that showed adequate reactivity for dc-ELISA. Before homogenization of vegetable samples, phosphoric acid was added (vegetable-10% phosphoric acid (2 : 1, w/v)) to block enzymatic decomposition of chlorothalonil. The use of phosphate buffer (100 mmol/L, pH 7.0) minimized the influence of phosphoric acid on competitive reaction in the dc-ELISA. Working range was 0.10 to 6.0 ng/mL in the optimized dc-ELISA. The recovery of chlorothalonil spiked in cucumber and eggplant was 97.1 to 125%. The results correlated well with those obtained by HPLC analysis. The dc-ELISA could rapidly determine chlorothalonil after a simple sample preparation procedure.

Modulatory effects of deltamethrin-exposure on the immune status, metabolism and oxidative stress in gilthead seabream (Sparus aurata L.).

Deltamethrin, a sintetic pyrethroid, is the insecticide that has been replacing recently to others like organochlorines, organophosphates and carbamates which are less toxic for birds and mammals, although, unfortunately, all of them are highly toxic to various non-targeted aquatic organisms including fish. In the present study, the consequences of the exposition of gilthead seabream (Sparus aurata L.) specimens to sublethal bath dose of deltamethrin (0.1 ppb) on organo-somatic indexes, immunity, seric metabolic parameters, oxidative stress and liver histology were determined after 1, 3, 7 and 14 days of exposure. Deltamethrin alters gilthead seabream immune status, the hepato-somatic index and various seric metabolic parameters since the first exposure day while important progressive deleterious morphological changes in liver were also observed. However, no statistically significant deviation was detected in the expression of oxidative stress-related genes whilst the expression of cytochrome P450 gene was up-regulated in head-kidney and liver of exposed fish. Overall, the present results indicate severe immunotoxicological and metabolic effects of deltamethrin in gilthead seabream, the species with the highest rate of production in Mediterranean aquaculture. In general, the values obtained for the tested parameters during the trial seem to indicate that specimens try to adapt to this adverse situation although the continuous presence of the toxic impede the hypothetic recovery of homoeostasis. The use of deltamethrin in the proximities of seabream farms should be carefully considered.

Development of an enzyme-linked immunosorbent assay for cyhalothrin.

In the present study, we obtained a specific monoclonal antibody (cross-reaction to analogues <5%) against cyhalothrin using two haptens. After 7 reaction steps, 3-cyano-[(cis)-3-(2-chloro-3, 3, 3-trifluoroethenyl-2, 2-dimethyl)-cyclopropane-carbonyloxy]-phenoxybenzyl propanoic acid was prepared with yield 35.9%. Four coating antigens and two immunogens were prepared. A heterologous enzyme-linked immunosorbent assay for cyhalothrin was established with the 50% inhibition concentration (IC50, 13.26 ± 1.23 ng mL(-1)) after optimizing various parameters including coating antigens, blocking agents, ionic strength, pH value and methanol concentration in the assay buffer. To evaluate the proposed immunoassay, spiked samples from river, tap water and drinking water at three levels (0.2, 1.0, 5.0 mg L(-1)) were tested after simple dilution. The mean recoveries ranged from 75.4% to 97.7% with coefficient of variation 5.1%-11.6%. The results from the above indicated the potencies of this ELISA in cyhalothrin analysis.

[Current contact allergens]. / Aktuelle Kontaktallergene.

Ever-changing exposure to contact allergens, partly due to statutory directives (e.g. nickel, chromate, methyldibromo glutaronitrile) or recommendations from industrial associations (e.g. hydroxyisohexyl 3-cyclohexene carboxaldehyde), requires on-going epidemiologic surveillance of contact allergy. In this paper, the current state with special focus in fragrances and preservatives is described on the basis of data of the Information Network of Departments of Dermatology (IVDK) of the year 2010. In 2010, 12,574 patients were patch tested in the dermatology departments belonging to the IVDK. Nickel is still the most frequent contact allergen. However the continuously improved EU nickel directive already has some beneficial effect; sensitization frequency in young women is dropping. In Germany, chromate-reduced cement has been in use now for several years, leading to a decline in chromate sensitization in brick-layers. Two fragrance mixes are part of the German baseline series; they are still relevant. The most important fragrances in these mixes still are oak moss absolute and hydroxyisohexyl 3-cyclohexene carboxaldehyde. However, in relation to these leading allergens, sensitization frequency to other fragrances contained in the mixes seems to be increasing. Among the preservatives, MCI/MI has not lost its importance as contact allergen, in contrast to MDBGN. Sources of MCI/MI sensitization obviously are increasingly found in occupational context. Methylisothiazolinone is a significant allergen in occupational settings, and less frequently in body care products.

Deltamethrin Increases Candida albicans infection susceptibility in mice.

Deltamethrin, an alpha-cyano type II synthetic pyrethroid insecticide, is used to control a wide range of insects on a variety of crops and vectors of diseases. Deltamethrin has been previously reported for its immunotoxic effects and therefore its exposure may affect the host resistance to infection and tumour challenge. Effect of exposure of deltamethrin on host resistance to Candida albicans infection was examined in Swiss albino mice. The objective of this study was to investigate the modulatory action of deltamethrin in C. albicans infected mice. The dose of deltamethrin was initially tested and selected from our previous study (18 mg/kg). Percentage of infection in deltamethrin treated animals increased faster when compared to that of the controls. Deltamethrin exposure along with C. albicans infection caused alteration of humoral immune response. The number of colony forming unit in liver and spleen were also found to be significantly increased in the treated group. The results from our present study suggest that deltamethrin exhibits an immunosuppressive effect and has a negative impact on host resistance to C. albicans infection.

Ethosome formulation of contact allergens may enhance patch test reactions in patients.

BACKGROUND: Ethosomes and liposomes are ultra-small vesicles capable of encapsulating drugs and cosmetic ingredients for topical use, thereby potentially increasing bioavailability and clinical efficacy. So far, few reports have suggested that formulation of cosmetic ingredients in vesicular carrier systems may increase the allergenicity potential. OBJECTIVES: To investigate the effect of ethosome formulation of isoeugenol and methyldibromo glutaronitrile on the elicitation response under patch test conditions and by repeated open applications. PATIENTS/MATERIALS/METHODS: A total of 27 volunteer patients with a previous positive patch test reaction to either isoeugenol or methyldibromo glutaronitrile were included in the study. In all patients, a serial dilution patch test was performed with the allergen in question formulated in ethosomes and in an ethanol/water solution. In addition, a repeated open application test (ROAT) was performed in a subset of 16 patients, and lag time until a positive response was recorded. RESULTS: Both contact allergens encapsulated in ethosomes showed significantly enhanced patch test reactions as compared with the allergen preparation in ethanol/water without ethosomes. No significant difference in the median lag time was recorded between preparations in the ROAT. CONCLUSIONS: Encapsulating potential contact allergens in ethosomes may increase the challenge response as compared with the same concentrations in an ethanol/water base without ethosomes.

Patch test dose-response study: polysensitized individuals do not express lower elicitation thresholds than single/double-sensitized individuals.

BACKGROUND: It is not known if reduced elicitation thresholds are evident among polysensitized individuals when using allergens to which the patients are already sensitized. Reduced elicitation thresholds may be an expression of increased reactivity in this patient group. OBJECTIVES: To examine and compare elicitation dose-response curves and elicitation thresholds in a polysensitized vs. a single/double-sensitized group for allergens to which the test subjects were already sensitized. PATIENTS/METHODS: Fifty-one patients (13 polysensitized and 38 single/double-sensitized) were patch tested with nickel sulphate, methyldibromo glutaronitrile (MDBGN) and p-phenylenediamine (PPD) in dilution series. The ratio between the doses eliciting a response in 50% of patients in the two groups was used as the measure for relative sensitivity. RESULTS: The dose-response curves of the polysensitized group for MDBGN and PPD were shifted to the right, and for nickel sulphate shifted to the left, compared with the single/double-sensitized group. The relative sensitivity for each of the three allergens and a combined relative sensitivity for all three allergens were not significantly different when comparing the polysensitized and single/double-sensitized groups. CONCLUSION: No increased sensitivity, in the form of distinct elicitation thresholds, could be demonstrated in polysensitized individuals compared with individuals with one or two contact allergies.

New aspects in allergic contact dermatitis.

PURPOSE OF REVIEW: To give selected new information on contact allergy and allergic contact dermatitis with focus on diagnostic procedures and pitfalls. RECENT FINDINGS: Recent studies dealing with common contact allergens have improved our understanding of the relationship between positive patch tests and the clinical interpretation and consequences for the patient. SUMMARY: Nickel allergy is still the most common contact allergy in Europe in spite of full implementation of the EU Nickel Directive in 2001. Contact allergens in cosmetics and topical drugs are another common cause of allergic contact dermatitis. The main culprits include fragrance chemicals, preservatives, and hair dyes. We are all more or less exposed to cosmetics and topical drugs on a daily basis. The labelling requirements given in the Cosmetics Directive is of great help in tracing the causative allergenic ingredients. Most of the components present in cosmetic products are also occurring in household and industrial products, often under other trade names. Patients with multiple contact allergies constitute a special problem because their quality of life is severely affected by the multitude of eliciting products in the environment. We still lack a good understanding of why these patients become so easily sensitized.

Development of an immunochromatographic assay for the rapid detection of bromoxynil in water.

A rapid immunochromatographic one-step strip test was developed to specifically determine bromoxynil in surface and drinking water by competitive inhibition with the nano colloidal gold-conjugated monoclonal antibody (mAb). Bromoxynil standard samples of 0.01-10 mg L(-1) in water were tested by this method and the visual limit was 0.06 mg L(-1). The assay only required 5 min and one-step by dispensing a drop of sample solution onto a strip. Parallel analysis of water samples with bromoxynil showed comparable results from one-step strip test and ELISA. Therefore, the one-step strip test is very useful as a screening method for qualitative detection of bromoxynil in water.

Toll-like receptor agonists stimulate human neutrophil migration via activation of mitogen-activated protein kinases.

Human neutrophil migratory responses to Toll-like receptor (TLR) agonists were studied using videomicroscopy. When challenged with lipopolysaccharide (LPS, TLR4 agonist) or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (P3CSK4, TLR2 agonist), neutrophils displayed enhanced motility, which was found to reflect increased random migration but not directed migration (chemotaxis). Enhanced neutrophil motility was detected within 10 min after stimulation with LPS or P3CSK4, and was sustained for more than 80 min. Stimulation of neutrophils with LPS or P3CSK4 resulted in the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), which preceded neutrophil migration. TLR-mediated neutrophil migration was strongly suppressed by pretreatment of cells with U0126 (MAPK/ERK kinase inhibitor) but not with U0124 (an inactive analogue of U0126) or SB203580 (a p38 MAPK inhibitor), and was almost completely abolished by pretreatment of cells with U0126 and SB203580 in combination. Randomly migrating neutrophils in response to LPS or P3CSK4 displayed directed migration when further challenged with gradient concentrations of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF). These findings indicate that TLR agonists stimulate human neutrophil migration via the activation of ERK and p38 MAPK, and FMLP- or PAF-induced neutrophil chemotaxis is not affected by the pre-exposure of cells to TLR agonists.

Comparison of three adjuvants used to produce polyclonal antibodies to veterinary drugs.

Two commercially available adjuvants, Gerbu LQ 3000 and Montanide ISA 50V, were assessed as potential replacements for Freund's adjuvant by evaluating their efficacy in the production of polyclonal antibodies to veterinary drugs in rabbits. The aim was to find an adjuvant that could produce a similar (or enhanced) immune response in the host animal without the undesirable side effects associated with Freund's complete and incomplete adjuvant. The assessment involved the examination of each injection site and the characterisation of the resultant antibodies with regards to antibody titre and sensitivity. It was found that the rabbits immunised with Gerbu adjuvant produced some of the most sensitive antibodies. However, titres were relatively low and adverse effects at injection sites were relatively common. Montanide adjuvant produced no adverse effects and the related antibodies were found to be of adequate sensitivity when compared to those from rabbits immunised with Freund's. It was concluded that Montanide ISA 50V could be considered as a suitable replacement to Freund's for the production of polyclonal antibodies, to low molecular weight compounds in rabbits.

Use of antigen mimics to produce specific antibodies to anti-coccidial drugs.

A range of polyclonal antibodies was successfully produced to the coccidiostatic drugs diclazuril and robenidine. Initial attempts to make immunogenic complexes of both drugs were ineffective due to difficulties encountered while trying to couple the compounds to large carrier proteins. Structural mimics, which could act as haptens for each drug, were sought and identified. The compounds identified were more open to chemical manipulation and were conjugated to carrier proteins to produce effective immunogens. The most sensitive antisera produced displayed IC50s of 1.5 ng/ml and 13 ng/ml for diclazuril and robenidine respectively. The antibody for diclazuril was shown to be specific, cross-reacting only with clazuril by 15%. The robenidine antibody displayed a low cross-reactivity of 1.2% to the compound used to produce the antibody.

Contact allergy epidemics and their controls.

Contact dermatitis can be severe and lead to sick leave as well as significant healthcare expenses. The aim of this review is to present the published knowledge on 6 historical epidemics of contact allergy to apply this knowledge on the prevention and control of future contact allergy epidemics. A historical review is performed on nickel, chromium, methyl-chloro-iso-thiazolinone/methyl-iso-thiazolinone, methyl-di-bromo glucaro-nitrile, formaldehyde, and para-phenylenediamine. The first cases of contact dermatitis are mostly occupational, whereas consumer cases appear later. There is often a latency period from the first cases are observed until an epidemic occurs, and the problem is recognized. Finally, no one seems to take responsibility of dealing with the situation, and there are no attempts of regulation until an epidemic is consolidated among consumers for many years. Steps should be taken to prevent contact allergy epidemics. It is essential that dermatologist, scientists, administrators, and consumers organize and structure known methods to accelerate the control of emerging contact allergens.