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Imidacloprid is the most widely used insecticide in agriculture and its intensive use over the last 30 years has caused a global concern due to its potentially toxic effects on the ecosystem. Considering the recent scientific interest in novel simple methods for imidacloprid analysis, we propose a label-free sensitive and specific localised surface plasmon resonance system for the detection of the insecticide based on 2D nanostructured metasurfaces with highly performing plasmonic properties. The specificity of the sensor proposed was achieved by covalent bio-functionalization of the metasurface using a smart and easy one-step procedure mediated by carbon disulphide. The biosensor produced was tested using a set of imidacloprid standard solutions showing a competitive limit of detection, lower than 1 ng mL -1 . Our novel nanosensing configuration represents a valid and reliable solution to realize low-cost portable POC tests as an alternative to the laborious and expensive methods traditionally used for insecticide detection.

Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC 50 values of 0.73 and 0.46 μg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH-π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.

Imidacloprid (IMD) is one of the most used pesticides worldwide as a systemic insecticide as well as for pest control and seed treatment. The toxic and potential carcinogenic character of IMD makes its monitoring of great relevance in the field of agriculture and environment, so sensitive methodologies for in field analysis are strongly required. In this context, we have developed a competitive immunoassay for the determination of IMD using specific monoclonal antibodies followed by electrochemical detection on screen-printed carbon electrodes (SPCE). The optimized immunosensor exhibited a good reproducibility (RSD of 9%) and a logarithmic response in the range 50-10 000 pM of IMD, with an estimated detection limit (LOD) of 24 pM, which was below the maximum levels allowed by the legislation. High-Performance Liquid Chromatography-Mass Spectrometry-Mass Spectrometry (HPLC-MSMS) and Enzyme-Linked Immunosorbent Assay (ELISA) analysis were also performed for comparison purposes, where the electrochemical immunosensor exhibited a wider range of response and a lower detection limit. Matrix effects below 6.5% were obtained using tap water samples. All these characteristics make our electrochemical immunosensor a valid and advantageous tool for the in field determination of IMD.

Immunoassays provide a high-throughput method for monitoring pesticides in foods and the environment. Due to easy generation and capable of being manipulated, chicken single-chain variable fragment (scFv) is attractive in the development of immunoassays for pesticides. Two scFvs (X1 and X2) against the insecticide carbaryl were generated from a chicken immunized with hapten C1 conjugated to keyhole limpet hemocyanin and fused with alkaline phosphatase (AP) to develop a rapid one-step enzyme-linked immunosorbent assay for this pesticide. X2-AP showed higher binding affinity to carbaryl than X1-AP. The X2-AP-based ELISA had a half-maximum signal inhibition concentration of 15 ng mL -1 and a limit of detection of 1.6 ng mL -1 . This assay showed negligible cross-reactivity with other carbamate pesticides (<0.1%) and low cross-reactivity with 1-naphthol (5%). The average recoveries of carbaryl spiked in soil, apple and pear samples by the one-step assay ranged from 90% to 114% and agreed well with those of high-performance liquid chromatography. The chicken scFv-based assay showed promise as a high-throughput screening tool for carbaryl in environmental and food matrices., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The insecticide fipronil can be metabolized to its sulfone in mammalian species. Two camel single-domain antibodies (VHHs) F1 and F6, selective to fipronil and fipronil-sulfone, respectively, were generated and used to develop enzyme linked immunosorbent assays (ELISAs) for the detection of the two compounds in the sera of black-tailed prairie dogs and rats. The limits of detection of fipronil and fipronil-sulfone in the rodent sera by the corresponding ELISAs were 10 and 30 ng mL -1 , and the linear ranges were 30-1000 and 75-2200 ng mL -1 . ELISAs showed a good recovery for fipronil and fipronil-sulfone cospiked in the control sera of the black-tailed prairie dogs (90-109%) and rats (93-106%). The VHH-based ELISAs detected fipronil and fipronil-sulfone in the sera of the rodents that received a repeated oral administration of fipronil. The average concentration of fipronil-sulfone was approximately 3.2-fold higher than fipronil in the prairie dog sera (1.15 vs 0.36 μg mL -1 ) and rat sera (1.77 vs 0.53 μg mL -1 ). ELISAs agreed well with a liquid chromatography-mass spectrometry method for the quantification of both fipronil and fipronil-sulfone in real serum samples. Fipronil-sulfone was identified as the predominant metabolite of fipronil in the black-tailed prairie dog and rat sera.

A monoclonal antibody (3A5) that can recognize thiacloprid was produced, and a linear 8-residue peptide phage library was constructed. Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library. A phage enzyme-linked immunosorbent assay (ELISA) was developed to detect thiacloprid using a phage-displayed peptide. Under the optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (IC10) of the developed phage ELISA were 8.3 and 0.7 μg/L, respectively. Compared with the conventional ELISA, the sensitivity was improved more than 3-fold. The cross-reactivity (CR) was less than 0.08% for the tested structural analogues and was regarded as negligible. The recoveries of thiacloprid ranged from 80.3% to 116.3% in environmental and agricultural samples, which conformed to the requirements for residue detection. The amount of thiacloprid detected by phage ELISA in the samples was significantly correlated with that detected by high-performance liquid chromatography. The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this study, the first mechanism-based monoclonal antibodies have been produced that recognize and differentiate diethoxy- and monoethoxyphosphorylated serine residues. Haptens were synthesized as the stable phosphonate form of phosphoserine esters to improve the immunoresponse. Following condensation with a glutaric anhydride to link the phosphoserine moieties to carrier protein, the hapten densities attached to bovine serum albumin and keyhole limpet henocyanin were determined by partial trypsin digestion and MALDI mass spectrometry, and confirmed using a fluorescent assay (FITC) to quantify unmodified lysine residues. The conjugation reactions were pH optimized to improve hapten density. Screening of subclones led to the identification of two monoclonal antibodies: (a) N257/25.11 that specifically recognizes (EtO)2P(O)-Ser as the phosphylated or inhibited form, and (b) N262/16 that recognizes (EtO)(HO)P(O)-Ser as the 'aged' form. Analysis of blood samples treated with paraoxon (EtO)2P(O)-OPhNO2 showed a concentration dependent recognition of the phosphylated form., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A label-free immunosensor based on SWNTs modified GC electrodes has been developed for the direct detection of paraoxon. Based on aryldiazonium salt chemistry, forest of SWNTs can be vertically aligned on mixed monolayers of aryldiazonium salt modified GC electrodes by C-C bonding, which provides an interface showing efficient electron transfer between biomolecules. PEG molecules were introduced to the interface to resist non-specific protein adsorption. Ferrocenedimethylamine (FDMA) was subsequently attached to the ends of SWNTs through the amide bonding followed by the attachment of epitope i.e., paraoxon hapten to which a paraoxon antibody would bind. This immunosensor shows good selectivity and high specificity to paraoxon, and is functional for the detection of paraoxon in both laboratory and field by a displacement assay. There is a linear relationship between electrochemical signal of FDMA and the concentration of paraoxon over the range of 2-2500 ppb with a lowest detected limit of 2 ppb in 0.1 M phosphate buffer at pH 7.0. The SWNTs based amperometric immunosensor provides an opportunity to develop the sensing system for on-site sensitive detection of a spectrum of insecticides., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5&#8211;15 ng/mL. The antibody showed high cross-reactivity (64%) to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

Objective: The objective is to investigate the effect of malaria control with insecticide-treated mosquito nets (ITNs) regarding possible higher mortality in children protected during early infancy, due to interference with immunity development, and to assess long-term effects on malaria prevalence and morbidity., Methods: Between 2000 and 2002, a birth cohort was enrolled in 41 villages of a malaria holoendemic area in north-western Burkina Faso. All neonates (n = 3387) were individually randomised to ITN protection from birth (group A) vs. ITN protection from age 6 months (group B). Primary outcome was all-cause mortality. In 2009, a survey took place in six sentinel villages, and in 2010, a census was conducted in all study villages., Results: After a median follow-up time of 8.3 years, 443/3387 (13.1%) children had migrated out of the area and 484/2944 (16.4%) had died, mostly at home. Long-term compliance with ITN protection was good. There were no differences in mortality between study groups (248 deaths in group A, 236 deaths in group B; rate ratio 1.05, 95% CI: 0.889-1.237, P = 0.574). The survey conducted briefly after the rainy season in 2009 showed that more than 80% of study children carried asexual malaria parasites and up to 20% had clinical malaria., Conclusion: Insecticide-treated mosquito net protection in early infancy is not a risk factor for mortality. Individual ITN protection does not sufficiently reduce malaria prevalence in high-transmission areas. Achieving universal ITN coverage remains a major challenge for malaria prevention in Africa., (© 2012 Blackwell Publishing Ltd.)

Background: Imidacloprid is one of the main neonicotinoid insecticides widely used in agriculture owing its broad spectrum of activity and low bioaccumulation. However, imidacloprid is toxic to honey bees and other beneficial organisms, and its residues may occur in environmental and food samples, posing a potential hazard to consumers. In this study the imidacloprid derivative bearing a three-atom length spacer was synthesized and coupled to carrier proteins. Highly sensitive and specific polyclonal antibodies against imidacloprid were successfully produced and the polyclonal antibody-based enzyme-linked immunosorbent assay (pAb-ELISA) was developed., Results: The ELISA standard curve was constructed within the concentration range 0.1-100 ng mL(-1). The IC(50) value for nine standard curves was in the range 1.2-3.0 ng mL(-1) and the limit of detection was 0.03-0.16 ng mL(-1). The sensitivity of the assay was one order of magnitude higher than that in most published papers. There was almost no cross-reactivity of the antibody with four structurally related compounds (acetamiprid, nicotine, clothianidin and nitenpyram) and six other compounds, indicating that the assay displays not only high sensitivity but also high specificity. No detectable imidacloprid was found in 11 collected environmental and food samples by the assay. For imidacloprid-spiked samples, acceptable recoveries of 73.4-94.4% and intra-assay coefficients of variation of 2.2-12.8% were obtained. The assay was also validated with high-performance liquid chromatography (HPLC) and a good correlation of ELISA with HPLC was achieved., Conclusion: The proposed ELISA provides a sensitive, specific, simple and cost-effective quantitative/screening method for detecting imidacloprid in environmental and food samples., (Copyright © 2011 Society of Chemical Industry.)

A single-chain variable fragment (ScFv) complementary DNA (cDNA) library against fenitrothion was constructed, and ScFvs specific for fenitrothion were selected by ribosome display from the library. After three rounds of ribosome display, the ScFv genes were cloned into Escherichia coli for expression. The expressed ScFvs of 160 clones were analyzed by indirect enzyme-linked immunosorbent assay (ELISA). Of these, 40 clones produced antibodies with relatively high activity against fenitrothion, and 3 were selected for Biacore and ELISA analysis. These 3 antibodies-ScFv-AF50, ScFv-AF93, and ScFv-AF132-had IC(50) values of 1.6, 3.4, and 2.2 ng/ml, respectively. Cross-reactivity with other organophosphorus (OP) pesticides was below 0.1% except for parathion-methyl (≤2.8%). The IC(50) values and cross-reactivity were lower than achieved previously with polyclonal or monoclonal antibodies against fenitrothion. The equilibrium dissociation constant (K(D)) values determined by Biacore analysis were 4.56×10(-10)M for ScFv-AF50, 1.42×10(-9)M for ScFv-AF93, and 2.66×10(-10)M for ScFv-AF132. These results demonstrate that the ribosome display has great potential in selection of ScFvs against pesticides. Recoveries of fenitrothion from fortified rice and cucumber were in the range 80.6 to 108%, indicating that the ELISAs with the isolated ScFvs can accurately determine fenitrothion in food samples after the simple and rapid extraction procedure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To produce high-affinity monoclonal antibodies against pesticide imidacloprid, we synthesized the haptens 1-[(6-Carboxylethylthio-3-pyridinyl) methyl] -N-nitro-imidazolidinimine (named as H1) and 1-[(6-Chloro-3-pyridinyl) methyl]-3-carboxylpropyl-N-nitro-2-imidazolidinimine (termed as H2). And then the haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA) for immunogen (H1-BSA) and coating antigen (H2-OVA) respectively by NHS ester method. BALB/c mice were immunized with H1-BSA conjugate. We obtained two hybridoma cell lines 2F11/A9 and 2G6/G12 secreting antibody specific for imidacloprid from the conventional hybridoma technology. The result showed that the subtypes of obtained monoclonal antibodies were IgG3 and IgG1, respectively, and the titers of ascites were up to 1:128 000. The indirect competitive ELISA indicated the IC50 values of 5.3 and 28.3 ng/mL with detection limits of 1.1 ng/mL and 7.7 ng/mL, respectively. Two monoclonal antibodies had no apparent cross reactivity with six analogous compounds. Thus, two prepared monoclonal antibodies had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of imidacloprid residue and laid a solid foundation for research and development of products for immunoassay.

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., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

The rhizomes and roots of Valeriana fauriei were extracted and the major essential oil composition and immunotoxicity effects were studied. The analyses were conducted by gas chromatography-mass spectroscopy (GC-MS) revealed that the essential oils of V. fauriei. The V. fauriei essential oil (VFEO) yield was 1.93%, and GC/MS analysis revealed that its major constituents were bornyl acetate (32.83%), terpinyl acetate (3.82%), bornyl isovalerate (2.11%), β-sesquiphellandrene (2.21%), sesquiterpene alcohol (7.32%), and cedrol (2.45%). The essential oil had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L with an LC(50) value of 30.44 ppm and an LC(90) value of 82.64 ppm. The results could be useful in search for newer, safer, and more effective natural immunotoxicity agents against Aedes aegypti L.

The leaves of Lindera obtusiloba Blume var. obtusiloba were extracted and the major essential oil composition and immunotoxicity effects were studied. The analyses were conducted by gas chromatography and mass spectroscopy (GC-MS) revealed that the essential oils of L. obtusiloba. The L. obtusiloba essential oil yield was 4.23%, and GC/MS analysis revealed that its major constituents were α-copaene (31.42%), β-caryophyllene (32.11%), α-humulene (4.12%), β-farnesene (4.15%), α- cadinene (3.21%) and Nerolidol (6.84%). The essential oil had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L with an LC(50) value of 24.32 ppm and an LC(90) value of 36.42 ppm.

The leaves of Zingiber officinale Roscoe were extracted and the major essential oil composition and immunotoxicity effects were studied. The analyses were conducted by gas chromatography and mass spectroscopy (GC-MS) revealed that the essential oils of Z. officinale leaves. The Z. officinale essential oil yield was 0.26%, and GC/MS analysis revealed that its major constituents were Camphene (5.26%), Phellandrene (6.58%), Zingiberene (36.48%), Geranial (4.32%), β-gurjunene (2.74%), and Citronellol β-sesguiphellandrene (12.31%). The essential oil had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L with an LC(50) value of 46.38 ppm and an LC(90) value of 84.32 ppm. Also, Camphene (≥95.0%), Phellandrene (≥95.0%), Zingiberene (≥95.0%), Geranial (≥95.0%), β-gurjunene (≥97.0%), and Citronellol (≥95.0%) were tested against the F21 laboratory strain of A. aegypti. Zingiberene (≥95.0%) and Citronellol (≥95.0%) have medium activity with an LC(50) value of 99.55 ppm and 141.45 ppm. This indicates that other major compounds may play a more important role in the toxicity of essential oil.

The flower parts of Paulownia coreana were extracted and the major essential oils composition and immunotoxicity effects were studied. The analyses were conducted by gas chromatography-mass spectroscopy (GC-MS) revealed that the essential oils of P. coreana. The P. coreana essential oil (PCEO) yield was 0.175%, and GC/MS analysis revealed that its major constituents were benzyl alcohol (13.72%), phenol, 3,4-dimethoxy-methyl ester (3.64%), phenol, 2-methoxy-3-(2-popenyl)-methyl ester (6.24%), 1,2,4-Trimethoxybenzene (8.32%), tricosane (3.28%), and pentacosane (3.26%). The essential oil had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L with an LC(50) value of 31.64 ppm and an LC(90) value of 56.43 ppm. 1,2,4-Trimethoxybenzene was the most toxic among the major components with an LC(50) value near 23.1 ppm. The results could be useful in search for newer, safer, and more effective natural immunotoxicity agents against A. aegypti.

Neonicotinoid insecticides control crop pests based on their action as agonists at the insect nicotinic acetylcholine receptor, which accepts chloropyridinyl- and chlorothiazolyl-analogs almost equally well. In some cases, these compounds have also been reported to enhance plant vigor and (a)biotic stress tolerance, independent of their insecticidal function. However, this mode of action has not been defined. Using Arabidopsis thaliana, we show that the neonicotinoid compounds, imidacloprid (IMI) and clothianidin (CLO), via their 6-chloropyridinyl-3-carboxylic acid and 2-chlorothiazolyl-5-carboxylic acid metabolites, respectively, induce salicylic acid (SA)-associated plant responses. SA is a phytohormone best known for its role in plant defense against pathogens and as an inducer of systemic acquired resistance; however, it can also modulate abiotic stress responses. These neonicotinoids effect a similar global transcriptional response to that of SA, including genes involved in (a)biotic stress response. Furthermore, similar to SA, IMI and CLO induce systemic acquired resistance, resulting in reduced growth of a powdery mildew pathogen. The action of CLO induces the endogenous synthesis of SA via the SA biosynthetic enzyme ICS1, with ICS1 required for CLO-induced accumulation of SA, expression of the SA marker PR1, and fully enhanced resistance to powdery mildew. In contrast, the action of IMI does not induce endogenous synthesis of SA. Instead, IMI is further bioactivated to 6-chloro-2-hydroxypyridinyl-3-carboxylic acid, which is shown here to be a potent inducer of PR1 and inhibitor of SA-sensitive enzymes. Thus, via different mechanisms, these chloropyridinyl- and chlorothiazolyl-neonicotinoids induce SA responses associated with enhanced stress tolerance.

This study aimed at developing competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) using a monoclonal antibody (mAb). Of the five EPN derivatives (haptens) prepared for use as an immunogen or as a competitor, two of them were used as the immunogen for the production of the mAbs. By using the antibody with the highest specificity and a coating antigen (hapten-OVA conjugate), a competitive indirect ELISA was developed, which showed an IC(50) of 2.9 ng/mL with a detection limit of 0.3 ng/mL. A competitive direct ELISA using a different antibody and an enzyme tracer was also developed, which showed an IC(50) of 0.6 ng/mL with a detection limit of 0.09 ng/mL. The mAbs in both assays showed negligible cross-reactivity with other organophosphorus pesticides. The recoveries of EPN from spiked samples determined by the developed ELISA ranged from 59 to 143%. Dilution of the samples improved the recovery. The assay performance of the present ELISAs based on the mAb was compared with that of the EPN ELISAs based on polyclonal antibodies (pAbs) that had been developed previously and was found to be better in dynamic response.

This paper reports for the first time the development of an immunoassay for the analysis of the miticide bromopropylate (BP). The chemical structure of the immunizing haptens was designed to maximize the recognition of the bis-bromophenyl group of BP. Thus, the assay uses polyclonal antibodies raised against 2,2-bis(4-bromophenyl)-N-2-hydroxyacetamide-butanoic acid (hapten 2) conjugated to keyhole limpet hemocyanin from horseshoe crab. A heterologous indirect competitive enzyme-linked immunosorbent assay (ELISA) has been developed that can detect BP down to 0.14 microg L(-1). The assay has been proven to tolerate a wide range of ionic strengths and pH values. Studies on the selectivity of this immunoassay have demonstrated a high recognition of related pesticides that contain a bis-halophenyl group in their structure. Other pesticides do not interfere in the analysis of BP using this immunochemical technique. Preliminary experiments have shown that BP can be directly analyzed in white wine samples down to 0.16 microg L(-1) without the necessity of a cleanup procedure prior to the ELISA.

A mouse hybrid hybridoma (tetradoma) was derived from fusing hybridomas producing monoclonal antibody to N-methylcarbamate pesticide carbofuran with hybridomas producing monoclonal antibody to organophosphorus pesticide Triazophos. The prepared tetradoma line (12C1 to 2H12) secreted hybrid immunoglobulin exhibiting parental and bispecific binding characteristics. The effect of relevant physicochemical factors on the immunoassay based on the 12C1 to 2H12 bispecific monoclonal antibody had been studied to optimize the ELISA performance. The developed immunoassay showed that the detection limit (I(20)) were 0.36 and 1.89 ng/mL for triazophos and carbofuran, respectively, without obvious cross-reactivity to other related compounds. Water samples spiked with triazophos at 0.5, 1, and 5 ng/mL or carbofuran at 5, 10, and 20 ng/mL were directly analyzed by the developed ELISA format. The mean recovery of triazophos and carbofuran were 108.1% and 107.5%, with variation coefficient of 15.9% and 17.7%, respectively.

Competitive enzyme-linked immunosorbent assays (ELISAs) in indirect and direct format were developed for the quantitative detection of the organophosphorus insecticide EPN. Five EPN derivatives (haptens) were synthesized and coupled to carrier proteins to use as an immunogen or as a competitor. Rabbits were immunized with two of the five haptens coupled to KLH for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin (OVA). Using the serum with the highest specificity and a coating antigen (hapten-OVA conjugate), an indirect (antigen-coated) ELISA was developed, which showed an IC(50) of 5.6 ng/mL with a detection limit of 0.2 ng/mL (20% inhibition). A direct (antibody-coated) ELISA using an enzyme tracer (hapten-enzyme conjugate) was also developed, which showed an IC(50) of 8.4 ng/mL with a detection limit of 0.9 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticide parathion-ethyl only in the direct ELISA. The recoveries of EPN from spiked samples determined by the indirect ELISAs were between 37 and 164%.

Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I50 of the assay ranged from 0.64 to 20.98 microg mL(-1) for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I50 and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL(-1), was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.

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.

A statistical approach for the analysis of complex samples by immunoassay is proposed in this article. Two enzyme-linked immunosorbent assays (ELISAs), one of them in the conjugate-coated format and the other in the antibody-coated format, were evaluated for their suitability to the analysis of mixtures of three organodithiophosphorus pesticides: azinphos-methyl, azinphos-ethyl and phosmet. It was found that the apparent affinity of the antibody to each analyte changed in the presence of a cross-reacting compound in the antibody-coated ELISA format, but not when the conjugate-coated ELISA format was used. The assays were thereafter applied to the analysis of mixtures of the three recognized pesticides. With the conjugate-coated ELISA format, accurate and precise determinations of mixtures could be performed if an azinphos-methyl standard curve was employed, with recoveries between 71% and 130% and with coefficients of variation lower than 12.7%. Neither accurate nor precise measurements could be accomplished with the enzyme immunoassay using the antibody-coated ELISA format, independently of the standard curve used. It is thought that the study presented here will have applicability in a variety of cases where the analytical goal is semiquantitative screening based on the total quantity of an unknown mixture of related compounds.

Pirimiphos-methyl (O-2-diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate: POM) is widely used organophosphorous (OP) insecticide as a grain protectant to control insects during storage. This study was carried out to assess the immunologic effects of POM in Balb/c mice after 28-day oral exposure. Three dose levels of POM (10, 60, or 120 mg/kg/day) were administered orally to mice for 4 weeks. At autopsy after 28-day exposure, there were significant decreases in relative spleen weight and splenic cellularity found at 120 mg POM, but body weight, relative thymic weight, thymic cellularity, and splenic and thymic subsets were not affected. T cell proliferation response induced by Con A was significantly decreased at all dosages though no statistical differences were observed in splenic B cell proliferation. Significant increases in the production of cytokines (IL-2, IL-4, IL-6, IFN-gamma, and IL-10) were evident on the whole, but the increase in production of inflammatory cytokines overwhelmed that of the T(H)1 cell suppressive cytokine (IL-10). The relative levels of three types of autoantibodies, anti-dsDNA, anti-histone, and antinuclear antibody (ANA) were dose-dependently decreased in serum. Oral exposure to POM induced a significant decrease in Immunoglobulin M production capability in Balb/c mice. This decrease in antibody production capability may result from disturbances in cytokine balance produced by splenic immune cells. These results show that POM may induce allergic responses by relatively enhancing T(H)2 development and additionally contribute to chronic inflammation by attracting macrophage by IFN-gamma.

To determine a possible role of glutathione (GSH) conjugation in 1,3-dibromopropane (1,3-DBP)-induced hepatotoxicity and immunotoxicity, female BALB/c mice were treated orally with 1,3-DBP. Based on the liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS) analyses, two forms of S-bromopropyl GSH were observed at m/z 427.9 and 429.9 in the positive ESI spectrum with a retention time of 5.29 and 5.23 min, respectively. Following single treatment of mice with 150, 300 or 600 mg/kg 1,3-DBP for 12 hr, the amount of S-bromopropyl GSH was detected maximally in liver homogenates at 600 mg/kg 1,3-DBP. Hepatic GSH levels were significantly decreased by treatment with 1,3-DBP. In a time course study, production of S-bromopropyl GSH rose maximally 6 hr after treatment and decreased gradually thereafter. The liver weights were significantly increased by treatment with 600 mg/kg 1,3-DBP. When mice were treated orally with 600 mg/kg 1,3-DBP for 12 hr, the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were increased by 365- and 83-fold. In addition, oral 1,3-DBP significantly suppressed the antibody response to a T-dependent antigen at 600 mg/kg 1,3-DBP. 1,3-DBP elevated hepatic levels of malondialdehyde and suppressed the activities of some hepatic enzymes involved in anti-oxidation. Taken together, the formation of GSH conjugate with 1,3-DBP may deplete cellular GSH and, subsequently, produce hepatotoxicity and immunotoxicity via damage to the cellular anti-oxidative system.

This study aimed at developing competitive enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus (OP) insecticide fenitrothion using a monoclonal antibody. The hapten used to obtain the antibody had an ideal structural feature that allowed minimal functional group sacrifice. By using the antibody and a coating antigen, a competitive indirect ELISA was developed, which showed an IC50 of 14 ng mL(-1) with a detection limit of 3.0 ng mL(-1). A competitive direct ELISA using an enzyme tracer was also developed, which showed an IC50 of 17 ng mL(-1) with a detection limit of 1.6 ng mL(-1). The antibodies in both assays showed negligible cross-reactivity with the metabolites of fenitrothion and other OP pesticides except with the insecticides parathion-methyl and parathion-ethyl. Recoveries of fenitrothion from fortified rice and lettuce samples were determined and the bias in the recovery values was rationalized by using the standard curves obtained in the matrix extract.

Enzyme-linked immunosorbent assay (ELISA) is the most convenient method of monitoring the occurrence of IgE antibodies specific for novel proteins in genetically modified (GM) foods. The levels of IgE specific for a recombinant protein, Cry1Ab, were determined using an ELISA method. A soluble form of the Cry1Ab protein purified from pCold1 vector-transformed Escherichia coli pTf16/BL21 was used as the ELISA coating antigen, and 1M NaCl was used as the washing buffer to remove IgE non-specifically bound to the coated antigen. Sera from 44 patients allergic to major food allergens were obtained, diluted 20-fold, tested, and found no identifiable IgE above background levels. We also tested sera from patients with corn allergy against whole extracts of non-GM and GM-corn (MON 810) using immunoblotting. The staining patterns were similar for the two types of corn. These results indicate that significant levels of IgE antibodies specific to Cry1Ab were not found in the sera of Japanese patients with food allergies.

A recombinant phage display library was generated using splenocyte mRNA isolated from a Balb/c mouse hyperimmunized with a hapten conjugate that mimicked the structure of methamidophos, one of the most acutely toxic organophosphate pesticides. Three recombinant single-chain variable fragment (scFv) antibodies with the highest specificity for methamidophos, termed 28D4, 29D0, and 36B2, were produced via a stringent selection protocol. In a competitive enzyme-linked immunosorbent assay, the IC50 values for 28D4, 29D0, and 36B2 were 46.25, 35.39, and 17.99 ng/mL, respectively. The cross-reactivity of the three scFv antibodies with other organophosphate pesticides was below 0.1% except for acephate (O,S-dimethyl acetylphosphoramidothioate). Nucleotide and deduced amino acid sequences indicated that the respective heavy chains and light chains of the three scFvs were involved in the distinctive VDJ segment rearrangements associated with somatic hypermutations during the process of several immunizations with higher dosages of immunogen. Taken together, these data constitute the first detailed description of an immunoassay that utilizes scFvs against the methamidophos, an analyte with a simple structure and low molecular mass (141 Da).

The botanical insecticide celangulin V (CA-V) is an insect digestive poison acting on midgut tissue of the target insect larvae. With the aim of localizing the receptor enacted by CA-V, monoclonal antibodies (MAbs) specific to the compound were developed. A hapten was synthesized by introducing a succinoyl into the CA-V structure and conjugated with three carrier proteins. From mice immunized with one conjugate, three MAbs were obtained with a potential capacity of detecting protein-bound residue forms of CA-V in the biological tissues. The oriental armyworm larvae ingested CA-V were examined by the technique of immuno-electron-microscopy (IEM) using the anti-CA-V MAb as the primary antibody and goat anti-mouse/IgG labeled with colloidal gold as the secondary antibody. Electron micrographs of the armyworm midgut tissues showed that the CA-V was associated with the midgut epithelia of the insects. These results demonstrated the existence of a receptor enacted by CA-V on the midgut cells of the oriental armyworm larvae.

The purified anti-carbofuran antibody was conjugated to carbonyl diimidazole (CDI)-activated Sepharose CL-4B to synthesize the immunosorbent for the immunoaffinity chromatographic (IAC) column specific to carbofuran. The conditions of IAC were optimized as follows: pH 7.2 phosphate buffer (PB) was used as equilibrium and adsorbent medium, and methanol-water (60:40, v/v) as eluent. The results showed that the dynamic column capacity was up to 1.58 mg/L bed volume. The efficiency of enrichment of IAC was more than 167 times when the initial concentration of carbofuran in a standard sample solution was lower than 2 microg/L. The spiked river water was cleaned up and enriched by IAC, and carbofuran in eluate was determined by enzyme linked immunosorbent assay (ELISA). The average recovery of carbofuran from river water was 89.8% with the relative standard deviation of 4.8% at the spiked level of 0.1 mg/L. Meanwhile the eluate was determined by high performance liquid chromatography (HPLC), the results from HPLC correlated well with those from ELISA. The IAC method of carbofuran was successfully established.

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.

Diazinon is a broad organophosphate insecticide used in agricultural and other treatments, resulting in widespread water contamination. The development of easy-to-use screening immunoanalytical methods is an interesting tool to study environmental pollution impact. Two novel strategies for diazinon hapten synthesis are addressed. One of them attaches the spacer arm to the oxygen atom of the diazinon aromatic ring. The other one retains the diazinon basic structure linking the spacer to an aromatic carbon. A total of eight diazinon haptens were synthesized, demonstrating that they are suitable for immunoreagent (protein conjugates and polyclonal antibodies) production. The optimized ELISA is based on conjugate-coated format and had a detection limit of 0.40 microg/L, showing little or no cross-reactivity to similar tested compounds. The immunoassays were used as a tool to quantify diazinon in natural waters. Results are in agreement with those given by GC-MS reference method. Mean recoveries ranging between 99% and 105% confirm the potential of our approach to determine diazinon in samples without purification or preconcentration steps, being applied as a screening method for field monitoring of diazinon in river waters.

Objective: To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies., Method: Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies., Results: Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 microg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 microg/mL and 1.03 microg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 microg/mL, and the linear working range and LOD were 10.91-11412.29 microg/mL and 3.42 microg/mL, respectively., Conclusion: Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.

A competitive enzyme-linked immunosorbent assay (ELISA) for fenoxycarb was adapted for quantitative detection of this insect growth regulator in various environmental, agricultural, food and biological matrices. Environmental samples were taken from soil and surface waters in Hungary. The ELISA enabled fenoxycarb detection in surface waters in the 1.1-125 ng ml(-1) concentration range without sample cleanup. In contrast, soil produced a strong matrix effect due to humic acids and other soil components. Several fruit homogenates and commercial fruit juices (eg apple, pear, grape) were analyzed by the ELISA. The assay was found to be suitable for analysis of fenoxycarb in fruit juices diluted 1:40. Biological samples included insect, fish and bovine tissues. The ELISA was applied to detect fenoxycarb in various biological matrices from larvae of the silkworm, Bombyx mori L. The assay proved useful for the analysis of haemolymph diluted 1:10 or at higher dilutions. Fat body and whole body homogenates, however, caused severe matrix effects. Fenoxycarb was detected in liver homogenates (diluted 1:40) from fish treated with various doses of fenoxycarb, and the concentrations determined correlated with the applied doses. The method was used to analyze spiked bovine urine samples diluted 1:10 or at greater dilutions. Fenoxycarb content determined by the ELISA in water and fruit juice samples was validated using GC-MS with solid-phase microextraction (SPME) sample preparation. The results of these studies demonstrated both the value and limitations of the assay when used for monitoring fenoxycarb in environmental, food and biological samples.

DDT (dichlorodiphenyltrichloroethane) reportedly induces cancer in animals, mimics estrogen activity, induces antiandrogen effects, and impairs Natural Killer (NK) cells and T lymphocyte responses. In this study, the authors attempted to determine associations of DDT, dichlorodiphenyldichloroethylene (DDE), and dichlorodiphenyldichloroethane (DDD) blood levels with several immune parameters in patients occupationally exposed to insecticides. The study subjects were 49 patients who worked as farmers or farmhands in the former German Democratic Republic and who had been occupationally exposed to insecticides for at least 6 mo; 80% of them had been exposed for more than 20 yr. Blood levels of DDT, DDE, DDD, 2,3,4,5,6-pentachlorophenol (PCP), polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), and gamma-hexachlorocyclohexanes (HCHs) were determined, and blood lymphocyte subpopulations, in vitro responses to mitogens or pooled allogeneic stimulator cells, plasma neopterin, and cytokine and soluble cytokine receptor levels were studied. The majority of patients were contaminated with more than 1 chemical--most commonly DDE, PCBs, and HCB. Linear-regression analysis showed that interleukin-4 (IL-4) plasma levels were associated with plasma levels of DDT 4.4 (p = .0001) and DDE 4.4 (p = .001). The data in this study suggest that PCBs, PCP, HCB, HCHs, DDE, and DDD suppress TH1 cytokines, such as IL-2 and interferon-gamma (IFN-gamma), and induce TH2 cytokines, such as IL-4. The authors hypothesized that clinical symptoms, such as the frequent infections reported by many patients, could be a consequence of these immunological abnormalities.

Twenty-nine individuals with chronic health complaints following exposure to chlorpyrifos were compared with 3 control groups (i.e., 1 positive and 2 negative) with respect to the following: (1) peripheral lymphocyte phenotypes; (2) autoantibodies (nucleic acids and nucleoproteins, parietal cell, brush border, mitochondria, smooth muscle, thyroid gland, and central nervous system/peripheral nervous system myelin); (3) mitogenesis to phytohemagglutinin and concanavillin. The data revealed an increase in CD26 expression, a decrease in percentage of CD5 phenotype, decreased mitogenesis in response to phytohemagglutinin and concanavillin, and an increased frequency of autoantibodies. The alterations in these peripheral blood markers were unaffected by medications, age, sex, or season. The authors concluded that chronic exposure to chlorpyrifos causes immunological changes.

Catalytic antibodies have been studied widely, but little is known about their applicability as therapeutic reagents in vivo. Here we report that carbaryl, a widely used broad-spectrum carbamate insecticide that is toxic to animals and humans, is hydrolyzed by polyclonal catalytic antibodies induced in vivo by a phosphate immunogen. To test the efficacy of the in vivo-induced polyclonal antibodies, we immunized mice with the phosphate immunogen and assayed their sensitivity to carbaryl by determining the ED(50) value, the dose that produces lowest-grade tremors in 50% of animals. We found that the ED(50) for immunized mice was 43% higher than that for nonimmunized mice and that this increase in ED(50) probably resulted from the hydrolysis of carbaryl by the catalytic antibodies in vivo. Our results suggest that polyclonal catalytic antibodies can be used as therapeutic reagents in vivo., (©2002 Elsevier Science (USA).)

Sensitive and selective enzyme-linked immunosorbent assays (ELISAs) in the immobilized antigen format were developed for fenoxycarb (1), an insect growth regulator (IGR). The parent molecule [ethyl 2-(4-phenoxyphenoxy)ethylcarbamate] was derivatized at several positions to obtain haptens (2-5) that were used to produce protein conjugates and rabbit polyclonal antisera. Amino derivatives of fenoxycarb at the terminal and internal rings (2 and 3, respectively) were linked to carrier proteins by azo coupling. Carboxyalkyl-spacer groups were attached to the ethyl group and the nitrogen atom of the target compound (1) to obtain haptens 4 and 5, respectively. Hapten-homologous ELISAs based on protein conjugates of compounds 2 and 4 determined fenoxycarb in the mid-ppb range (IC(50), 102 and 95 ppb, respectively). A more sensitive hapten-heterologous ELISA (IC(50), 17 ppb; detection limit 0.5 ppb) involved the antiserum raised against a conjugate of hapten 2 and the plate-coating antigen obtained from compound 3. These assays displayed no significant interferences with photodegradation products of fenoxycarb, the IGRs methoprene and pyriproxyfen, and a variety of pesticides including the pyrethroids fenvalerate and cypermethryn, the phenoxyacetic acid herbicide 2,4-D, DDT, and the nitrodiphenyl ether herbicides acifluorfen and fluorodifen.

Toxaphene, dissolved in glycerol/corn oil, was administered at 0.1, 0.4 or 0.8 mg/kg body weight/day in gelatin capsules to groups of 10 young adult female cynomolgus monkeys (Macaca fascicularis), while a group of five male monkeys (Macaca fascicularis) received 0.8 mg/kg body weight/day. Control male (a group of five) and female (a group of 10) monkeys ingested the glycerol/corn oil vehicle only. Treatment continued for 75 weeks. Testing for immune effects was initiated at 33 weeks of treatment. Immunization was initiated at 44 weeks of treatment. Pairwise comparisons between each of the treated female groups to the control indicated that the mean primary (post-immunization weeks 1-4) and secondary (post-immunization weeks 5-8) anti-SRBC IgM responses were significantly reduced at the 0.4 and 0.8 mg/kg body weight/day doses compared to the control (P< or =0.05). The mean primary (post-immunization weeks 1-4) anti-SRBC IgG response was significantly reduced compared to the control (P< or =0.05), while the secondary (post-immunization weeks 5-8) anti-SRBC IgG was not significantly affected by treatment (P>0.05). The mean anti-tetanus toxoid IgG response in the 0.8 mg/kg body weight/day dose group The mean primary anti-SRBC (IgM) response in the treated males was significantly different from the control (P<0.05), while the primary anti-SRBC IgG response was not affected by treatment. The mean absolute B-lymphocyte numbers in the female group administered 0.8 mg/kg of toxaphene was significantly reduced compared to the control (P< or =0.05). All other parameters including the natural killer cell activity, the delayed-type hypersensitivity response, the lymphoproliferative response of peripheral blood leukocytes to the mitogens Con A and PWM and the serum cortisol levels were not affected significantly by treatment (P>0.05). The no-observed-adverse-effect level (NOAEL) for the female monkeys based on the toxaphene effects on humoral immunity was 0.1 mg/kg body weight/day.

The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput.

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).

For determination of whether plasma 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) pesticide levels (< or =1-32 ppb) are associated with immune suppression or DNA damage in lymphocytes, 302 individuals residing in Moore County, North Carolina, in 1994-1996 provided a blood specimen, underwent a skin test, and answered a questionnaire concerning factors affecting plasma organochlorine pesticide levels and the immune system. The blood specimens were analyzed for levels of plasma DDE (a metabolite of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane), numbers and types of blood cells, immunoglobulin levels, mitogen-induced lymphoproliferative activity, and lymphocyte micronuclei. When DDE levels were categorized as 1 or less, more than 1 to 2, more than 2 to 4.3, more than 4.3 to 7.6, and more than 7.6 ppb, individuals with higher plasma DDE levels had lowered mitogen-induced lymphoproliferative activity (concanavalin A, range: 74,218 dropping to 55,880 counts per minute, p = 0.03) and modestly increased total lymphocytes (range: 2.0-2.3 x 10(3)/microl, p = 0.05) and immunoglobulin A levels (range: 210-252 mg/dl, p = 0.04). There were no consistent differences in response to the skin tests by plasma DDE levels. Plasma DDE levels were not associated with a higher frequency of micronuclei. The authors conclude that relatively low levels of plasma DDE are associated with statistically significant changes in immune markers, although the magnitude of the effects are of uncertain clinical importance.

Monoclonal antibodies (MAbs) to the pesticide propoxur were produced from haptens with carboxylic spacer arms of different lengths introduced at the carbamate group of the analyte structure. MAbs were subsequently characterized in the conjugate-coated format using these immunizing haptens and newly synthesized compounds as homologous and heterologous assay conjugates, respectively. Appropriate combinations of immunoreagents resulted in competitive enzyme immunoassays (ELISA) with I(50) values in the low nanomolar range (6.5-17.9 nM). A modification of the conjugate-coated format consisting of the simultaneous incubation of the MAb and the peroxidase-labeled secondary antibody in the presence of the analyte resulted in an assay with an I(50) value of 4.4 nM. This one-step conjugate-coated ELISA format is as simple and fast as the antibody-coated format but without the need of synthesizing enzyme-hapten conjugates. Major N-methylcarbamate pesticides were not recognized by the MAb. This immunoassay should reasonably allow the rapid, low-cost, and sensitive determination of propoxur in food, soils, and the environment at levels of regulatory and practical importance.

A rabbit polyclonal antiserum and two murine monoclonal antibodies recognizing the organophosphorus pesticide chlorpyrifos-ethyl were produced. The two hybridoma cell lines were then used as sources of immunoglobulin genes for the generation of recombinant scFv antibodies in Escherichia coli. The two scFvs showed either similar or improved limits of detection in an ELISA when compared with the monoclonal antibodies. Cross-reactivity studies showed that all of the antibodies were specific toward the chlorinated aromatic ring. Furthermore, scFv gene sequences were linked directly to sequences coding for either a c-Myc tag, a His-tag, or alkaline phosphatase. The fusion products generated were functional, and their properties were determined. The problems associated with producing scFvs and scFv derivatives for detection of pesticide residues from hybridoma are addressed and discussed.