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.

Isolation of broad-specificity domain antibody from phage library for development of pyrethroid immunoassay.

An antibody to phenoxybenzoic acid (PBA), the conserved chemical region of pyrethroids, was developed using a domain antibody (DAB) library to enable pyrethroid detection in agricultural products. The DAB library, constructed without animal immunization and based on a human VH framework, displayed repertoires on filamentous bacteriophage. After four rounds of panning, we obtained five domain antibodies that are capable of binding to PBA. Antibody A3 has strong identification capability to cypermethrin, ß-cypermethrin, and fenvalerate. The antibody A3 was used to develop an enzyme-linked immunosorbent assay (ELISA). The IC50 values were 2.586, 1.814, and 2.251 µg/ml for cypermethrin, ß-cypermethrin, and fenvalerate, respectively. The assay shows weak competition with flucythrinate but shows no competition with fenpropathrin, deltamethrin, and permethrin. The developed ELISA process was successfully applied to fortified Chinese cabbage samples, with the recoveries of cypermethrin, ß-cypermethrin, and fenvalerate ranging from 84.4 to 112.3%. We developed an immunoassay to detect pyrethroids depending on the domain antibody library, which overcomes the limitation of requiring protein antigen to immunize animals raising antibody.

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.

Prenatal exposure to pesticide ingredient piperonyl butoxide and childhood cough in an urban cohort.

RATIONALE: Previously we reported that airborne concentrations of cis-permethrin, but not trans-permethrin, measured during pregnancy in an inner city pediatric cohort was associated with cough by age 5. However, the effect of subsequent exposures to both permethrins during early childhood, and to piperonyl butoxide (PBO, a synergist for residential pyrethroid insecticides) remains to be elucidated. We hypothesized that prenatal and age 5-6 year measures of PBO and permethrins would be associated with cough at age 5-6 years in this cohort. Further, we explored the associations between these pesticide measures and wheeze, asthma, seroatopy, and fractional exhaled nitric oxide (FeNO). METHODS: PBO and permethrins were measured in personal air during the third trimester of pregnancy and indoor residential air at age 5-6 years (n=224). Health outcome questionnaires were administered to the mothers of 5-6 year old children. Indoor allergen specific and total immunoglobulin (Ig) E production was measured from sera collected at age 5, and FeNO was measured at 5-6 years. The hypotheses were tested using regression models adjusting for common confounders. RESULTS: Noninfectious cough was reported among 14% of children at age 5-6 years. Measures of prenatal PBO, but not age 5-6 year PBO or permethrins, increased the odds of cough [OR (95% CI): 1.27 (1.09-1.48), p<0.01; n=217]. No significant associations were found for other measured health outcomes. CONCLUSIONS: Prenatal PBO exposure was associated with childhood cough. It is unclear whether the observed effect is due mainly to PBO itself or residential pyrethroids of which PBO is an indicator.

Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay.

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC(50) of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC(50) = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.

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.

Class-specific immunoaffinity monolith for efficient on-line clean-up of pyrethroids followed by high-performance liquid chromatography analysis.

A class-specific monolithic immunoaffinity column was developed for on-line clean-up of pyrethroid insecticides in complex samples. Deltamethrin was oxidized with ozone to generate the hapten of (RS)-alpha-cyano-3-phenoxybenzyl (RS)-cis,trans-2,2-dimethyl-3-formyl-cyclopropane carboxylate. Class-specific antibodies against pyrethroids were produced using the conjugate of above hapten with bovine serum albumin as the immunogen. Poly(ethylene dimethacrylate-glycidyl methacrylate) monolith was synthesized in a 50mmx4.6mm i.d. stainless steel cartridge with two auxiliary pipette tips. The polymerization method was proved to be economic and reproducible. Antibodies against pyrethroids were covalently immobilized onto the monolithic support via Schiff base reaction. With a column-switching valve system, the immunoaffinity monolith (IAM) could be readily adapted to the reversed-phase high-performance liquid chromatography (HPLC) system. Under the optimum loading, washing and eluting conditions, the IAM specifically retained deltamethrin, flumethrin, flucythrinate and cis/trans permethrin, which were further baseline separated by C18 column using acetonitrile-water (83:17, V/V) as the mobile phase at a flow rate of 1.0mL/min. The established system provides a highly efficient approach for high-throughput on-line clean-up of pyrethroid in various samples.

Isolation of single chain variable fragments against six esters of pyrethrins by subtractive phage display.

Recombinant antibodies (rAbs) are a new diagnostic test for immulogical detection. To date, there are no reports about anti-pyrethrins rAbs. Here we describe the generation of monomeric and dimeric single chain variable fragments (scFvs) with affinity for six esters of pyrethrins using a subtractive phage display technology. First, scFv libraries with long-linker (Ger(4)Ser)(3) and short-linker (Ger(4)Ser) were established to contain 1.04 x 10(7) or 6.07 x 10(6) transformants. After four rounds of panning, phage ELISA demonstrated that three clones (E2, F2, and H7) showed higher affinity from the long-linker library, and clones (h6, a5) exhibited better antibody activity to pyrethrin I and II from the short-linker library. The scFv candidates were sequenced to identify the specific antibody response against pyrethrins. Isolated scFvs constitute valuable tools for real-time detection of pyrethrins. In addition, the subtractive phage display provides a simple approach for isolation of scFvs.

Phage-borne peptidomimetics accelerate the development of polyclonal antibody-based heterologous immunoassays for the detection of pesticide metabolites.

Competitive immunoassays for the detection of small analytes, such as pesticides and their metabolites, use haptens that compete with the target compounds for binding to the antibody. This competing hapten can be either the same as the immunizing hapten (homologous assay) or structurally modified mimics of the immunizing hapten (heterologous assay). Polyclonal antibody-based heterologous immunoassays have shown superior sensitivities to homologous ones, butthe synthesis of heterologous haptens may be time-consuming, requiring expertise in synthetic chemistry. In this work we demonstrate that phage display peptide libraries can be used as a source of phage-borne peptidomimetics to facilitate the development of sensitive heterologous assays. Different strategies for the isolation of these peptides were explored using two metabolites of pyrethroid insecticides. The sensitivities of the best competitive phage heterologous enzyme-linked immunosorbent assays were 13 fold and 100 fold better than the homologous assay, for the glycine conjugate of trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid and 3-phenoxybenzoic acid, respectively. The phage particles were highly versatile as tracer reagents, allowing the use of enzymatic, chemiluminescent, or immuno-polymerase chain reaction detection. The data presented here shows a new systematic procedure that enables the fast generation of several competing haptens for the rapid development of sensitive heterologous immunoassays.

Cutaneous contact urticaria to pyrethrum-real?, common?, or not documented?: an evidence-based approach.

Reports suggest that pyrethrum, the insecticidally active extract from Chrysanthemum cinerariifolium, can induce Type I hypersensitivity reactions in humans. Using knowledge of pyrethrum chemistry and an evidence-based analysis of literature, whether current refined pyrethrum induces and/or elicits skin manifestations of contact urticaria was assessed. Current extraction and refinement techniques suggest that refined pyrethrum lacks the presence of significant, if any, proteins speculated to induce Type I hypersensitivity. Our interpretation suggests that no reports of Type I reactions presented in the literature fulfill the criteria for immunologic contact urticaria. Future patient testing with current commercial material should clarify its Type I immunologic potential, if any.

Pyrethrum allergic contact dermatitis in humans--real?, common?, or not documented? An evidence-based approach.

Pyrethrum has been reported to produce allergic contact dermatitis in humans. Moreover, it has been speculated that cross reactions occur in ragweed-sensitized people. This review presents the botany, contemporary chemistry, and case reports of alleged allergic contact dermatitis. Our interpretation suggests that the evidence presented in literature does not show that allergic contact dermatitis results from exposure to pyrethrum. Similarly, the data do not suggest cross reactions in ragweed-sensitized people. Changes in the chemical composition of the refined pyrethrins suggest the putative sensitizer is present at a lower level in today's refined extracts than in ground pyrethrum flowers or the extracts used earlier.

Haemato-biochemical and immuno-pathophysiological effects of chronic toxicity with synthetic pyrethroid, organophosphate and chlorinated pesticides in broiler chicks.

Haemato- biochemical and immuno-pathophysiological changes following feeding of broiler chicks with 20 ppm fenvalerate (synthetic pyrethroid, SP), 2 ppm monocrotophos (organophosphate, OP) and 2 ppm endosulfan (chlorinated hydrocarbon, CH) were studied. Four groups of broiler birds (30 each) were fed poultry mash without (control) or mixed with pesticides for 8 weeks. Blood glucose, serum globulin and acetyl cholinesterase (AChE) activity level were decreased (P<0.01) in all treated groups compared to control, but not the serum albumin and BUN. The total ATPase activity was enhanced (P<0.01) in fenvalerate and monocrotophos than birds in control group. Body weight, total erythrocyte count, packed cell volume, haemoglobin, eosinophil and monocyte count did not show any changes. Total leucocytes and T-lymphocyte count was lower (P<0.01) in all treated groups as compared to control group. B-cell count (P<0.01), mean 2-4-dinitrofluorobenzene (DNFB) dermal sensitivity score and splenic indices from graft vs. host reaction (P<0.05) were decreased in fenvalarate and endosulfan but the values for monocrotophos were intermediate between control and other treated groups. Pesticide intoxication reduced nitroblue tetrazolium (NBT) positive cells (active splenic macrophages) (P<0.05) and spleen weight (P<0.01). Whereas bursal weight was reduced only with endosulfan, thymic weight was reduced on endosulfan and fenvalerate-treated feed. Microscopic examination of these organs further revealed atrophy/hypoplasia, decrease in the size of follicles with depletion of lymphocytes and haemorrhages in thymus. The study concludes that the chronic exposure of chicks to small amount of SP, OP and CH pesticide leads to deleterious effects on metabolism and immune system of birds.

Hapten and antibody production for a sensitive immunoassay determining a human urinary metabolite of the pyrethroid insecticide permethrin.

Permethrin is the most popular synthetic pyrethroid insecticide in agriculture and public health. For the development of the enzyme-linked immunosorbent assay (ELISA) to evaluate human exposure to permethrin, the glycine conjugate (DCCA-glycine) of a major metabolite, cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was established as the target analyte. Four different types of the cis- and trans-isomers of immunizing haptens were synthesized as follows: N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine (hapten 3), N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)-4-amino-l-phenylalanine (hapten 5), N-(N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine)amino-6-(2,4-dinitrophenyl)aminohexanoic acid (hapten 9), and N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine-4-oxobutanoic acid (hapten 24). Sixteen polyclonal antibodies produced against each cis- or trans-hapten-thyroglobulin conjugate as immunogens were screened against numerous hapten-bovine serum albumin conjugates as coating antigens. Six ELISAs with both a heterologous hapten structure and a heterologous hapten configuration (cis/trans or trans/cis) between antibody and coating antigen showed a high sensitivity for the target analyte. The IC50 was 1.3, 2.1, and 2.2 microg/L for the trans-target analyte and 0.4, 2.3, and 2.8 microg/L for the cis-target analyte. The immunizing haptens, except for hapten 5, provided the target specific antibodies. Molecular modeling of the haptens supported the selection of reasonable immunizing haptens that best mimicked the target analyte. Hapten 5 was suitable as a coating antigen rather than as an immunogen since it had a different geometry. Very low cross-reactivities were measured to permethrin, its free metabolite (DCCA), PBA-glycine conjugate, and glycine. The ELISA will be optimized for the detection of total cis/trans-DCCA-glycine in human urine samples.

Fluorescence quenching competitive immunoassay in micro droplets.

A fluorescence quenching competitive immunoassay in micro droplets was applied to the sensitive detection of the pyrethroid insecticide, esfenvalerate. Laser induced fluorescence from rhodamine dye was used as a marker. The competitive immunoreaction was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-microm diameter orifice. Fluorescence that was emitted from the droplets was detected by a 1/8 m imaging spectrograph with a 512 x 512 thermoelectrically cooled, charged-coupled device camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased. The reduction in emission was due to a strong quenching effect that arises from the interaction between the protein and rhodamine molecules following the antigen-antibody reaction. When a sample of esfenvalerate was added to the droplets, the release of the conjugated rhodamine from the antigen-antibody complex allowed the fluorescence signal to recover. An assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. A very small mass of analyte could be detected with this method. A sample of river water was used to gauge the impact of matrix effects and was shown to give rise to negligible interference with the immunoassay.

Enzyme-linked immunosorbent assay for the pyrethroid deltamethrin.

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of deltamethrin was developed. Two haptens, cyano[3-(4-aminophenoxy)phenyl]methyl 1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylate and 3-[(+/-)-cyano[1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropan ecarbonyloxy]methyl]phenoxyacetic acid, were synthesized and conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for deltamethrin was optimized and characterized. The I(50) for deltamethrin was 17.5 +/- 3.6 microg/L, and the lower detection limit was 1.1 +/- 0.5 microg/L. This ELISA assay had relatively low cross-reactivities with other major pyrethroids, such as permethrin, phenothrin, bioresmethrin, cyfluthrin, and cypermethrin. Methanol was found to be the best organic cosolvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was used for river water samples. River water samples fortified with deltamethrin were analyzed according to this method. Good recoveries and correlation with spike levels were observed.

A monoclonal antibody-based ELISA for the analysis of the insecticide flucythrinate in environmental and crop samples.

A competitive enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of the insecticide flucythrinate in environmental and food samples. Two types of haptens, the acid moiety that is the hydrolyzed product of flucythrinate, and the carboxylated propyl derivative of the alcohol moiety, were used to prepare monoclonal antibodies (MAbs). Five MAbs, which raised against the former hapten, were reactive with flucythrinate. Among them, MAb F1A27-4 showed the highest activity toward flucythrinate, and did not cross-react with other pyrethroids such as cycloprothrin, fenvalerate, fluvalinate, etofenprox and silafluofen. The assay conditions of indirect competitive ELISA with MAb F1A27-4 were studied to optimize the detection of flucythrinate in environmental and food samples. Incubation at 4 degrees C in the assay buffer, pH 8, with 300 mM sodium chloride improved the sensitivity. The addition of rabbit serum albumin or rabbit antiserum and the presence of 50 ml litre-1 of methanol reduced matrix effects of the samples. Under optimized conditions, the ELISA detected flucythrinate spiked in water, soil, and extracts of apple and tea samples down to 10 mg litre-1, 0.2 mg litre-1, 0.3 mg litre-1 and 0.3 mg litre-1, respectively. The mean recovery and CV ranged from 91% to 120% and from 5% to 12%, respectively. The ELISA results in apple samples correlated well with those from LC-MS analysis (r2 = 0.99, n = 12).

Effects of methanol and temperature on enzyme immunoassay with monoclonal antibodies specific to the insecticide etofenprox.

We examined the effects of methanol and temperature on the reactivity of monoclonal antibodies specific to the insecticide etofenprox. When the antigen-antibody reaction was done at 4 degrees C in 10% methanol, the sensitivity in the enzyme immunoassay with each antibody was more than 10-fold higher than that measured at 37 degrees C. Although in 10% methanol one of the antibodies reacted equally with both etofenprox and the carbonate-derivative of etofenprox, in 50% methanol the antibody reacted with etofenprox, but not with the derivative.

Effectiveness of polyclonal and monoclonal antibodies prepared for an immunoassay of the etofenprox insecticide.

Two polyclonal antibodies and three monoclonal antibodies specific to the etofenprox insecticide were prepared from rabbits and mice, respectively. The monoclonal antibodies were more reactive with etofenprox than the polyclonal antibodies by C-ELISA. Monoclonal antibody cl 205-65 was found to be the most tolerant to methanol, most highly reactive and most specific to etofenprox among the antibodies tested.