LSPR immuno-sensing based on iso-Y nanopillars for highly sensitive and specific imidacloprid detection.

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.

Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody.

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 IC50 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.

A monoclonal antibody-based immunosensor for the electrochemical detection of imidacloprid pesticide.

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.

One-step immunoassay for the insecticide carbaryl using a chicken single-chain variable fragment (scFv) fused to alkaline phosphatase.

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.

Quantitative Detection of Fipronil and Fipronil-Sulfone in Sera of Black-Tailed Prairie Dogs and Rats after Oral Exposure to Fipronil by Camel Single-Domain Antibody-Based Immunoassays.

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.

Immunodetection of the toxic portion of Vip3A reveals differential temporal regulation of its secretion among Bacillus thuringiensis strains.

AIMS: To devise a protocol for heterologous expression and purification of a partial toxic portion of the Bacillus thuringiensis (Bt) vegetative insecticidal protein Vip3A and using it as an antigen for anti-Vip3A polyclonal antibody development. Also, to evaluate the regulation of Vip3A secretion into culture supernatants (SNs) of different Bt strains based on this antibody. METHODS AND RESULTS: A primer pair was designed to amplify partially the toxic portion of the vip3A gene from the HD125 strain. The amplicon was cloned in expressing vector to produce a ~35 kDa peptide, which was HPLC-purified prior to rabbit immunizations. The serum containing the polyclonal anti-Vip3A antibody demonstrated a detection sensitivity of 0·4 ng mm-2 for the antigen in slot-blot experiments. Seven Bt strains from different origins were assessed regarding their temporal secretion of Vip3A toxin. ELISA results showed a strain-specific temporal regulation of Vip3A secretion in culture for the temperate isolates, with no detection of the toxin for the tropical strains, even when the presence of the gene was confirmed by PCR and sequencing. CONCLUSIONS: Conformational variation in the toxic portion of Vip3A may explain lack of its detection in the tropical strains. Isolates from the same subspecies display physiological variability in proteins' secretion into culture SNs, which can affect screening procedures for more effective strains/toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Immunoassays based on the developed anti-Vip3A antibody can be useful in a variety of basic studies. This method can be also coupled with toxicity assays on target insects, for more efficient screening methods of novel Bt strains/toxins with biocontrol applicability.

Haemocoel injection of PirA1B1 to Galleria mellonella larvae leads to disruption of the haemocyte immune functions.

The bacterium Photorhabdus luminescens produces a number of insecticidal proteins to kill its larval prey. In this study, we cloned the gene coding for a binary toxin PirA1B1 and purified the recombinant protein using affinity chromatography combined with desalination technology. Furthermore, the cytotoxicity of the recombinant protein against the haemocytes of Galleria mellonella larvae was investigated. We found that the protein had haemocoel insecticidal activity against G. mellonella with an LD50 of 131.5 ng/larva. Intrahaemocoelic injection of PirA1B1 into G. mellonella resulted in significant decreases in haemocyte number and phagocytic ability. In in vitro experiments, PirA1B1 inhibited the spreading behaviour of the haemocytes of G. mellonella larvae and even caused haemocyte degeneration. Fluorescence microscope analysis and visualization of haemocyte F-actin stained with phalloidin-FITC showed that the PirA1B1 toxin disrupted the organization of the haemocyte cytoskeleton. Our results demonstrated that the PirA1B1 toxin disarmed the insect cellular immune system.

Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide.

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.

Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.

The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody.

An electrochemical immunosensor based on interdigitated array microelectrode for the detection of chlorpyrifos.

An electrochemical immunosensor based on interdigitated array microelectrodes (IDAMs) was developed for sensitive, specific and rapid detection of chlorpyrifos. Anti-chlorpyrifos monoclonal antibodies were orientedly immobilized onto the gold microelectrode surface through protein A. Chlorpyrifos were then captured by the immobilized antibody, resulting in an impedance change in the IDAMs surface. Electrochemical impedance spectroscopy was used in conjunction with the fabricated sensor to detect chlorpyrifos. Under optimum conditions, the impedance value change of chlorpyrifos was proportional to its concentrations in the range of 10(0)-10(5) ng/mL. The detection limit was found to be 0.014 ng/mL for chlorpyrifos. The proposed chlorpyrifos immunosensor could be used as a screening method in pesticide determination for the analysis of environmental, agricultural and pharmaceutical samples due to its rapidity, sensitivity and low cost.

Detection of Bacillus thuringiensis Cry1Ab protein based on surface plasmon resonance immunosensor.

Two novel surface plasmon resonance immunosensors were fabricated for detection of the Bacillus thuringiensis Cry1Ab protein and to demonstrate their performance in analyzing Cry1Ab protein in crop samples. Sensor 2 was modified by 1,6-hexanedithiol, Au/Ag alloy nanoparticles, 3-mercaptopropionic acid, and protein A (or not [sensor 1]), with Cry1Ab monoclonal antibody. As a result, both of the immunosensors exhibited satisfactory linear responses in the Cry1Ab protein concentration ranges of 10 to 500ngml(-1) and 8 to 1000ngml(-1), and the detection limits were 5.0 and 4.8ngml(-1), respectively. The immunosensors possessed good specificity and acceptable reproducibility. In addition, crop samples could be analyzed after a simple treatment. The transgenic crops could be easily identified from the conventional ones by the two immunosensors.

Preparation and characterization of diethoxy- and monoethoxy phosphylated ('aged') serine haptens and use in the production of monoclonal antibodies.

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.

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.

Towards the fabrication of a label-free amperometric immunosensor using SWNTs for direct detection of paraoxon.

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.

An insecticidal protein from Xenorhabdus ehlersii stimulates the innate immune response in Galleria mellonella.

The bacteria Xenorhabdus spp. are entomopathogenic symbionts that can produce several toxic proteins that interfere with the immune system of insects. Recently, we purified the insecticidal protein XeGroEL from Xenorhabdus ehlersii and discovered that injection of XeGroEL into larvae of Galleria mellonella triggers strong immune responses. In this study, we determined the level of induction of several immune-responsive proteins that were secreted into the hemolymph using comparative proteomic analyses of hemolymph proteins from XeGroEL-challenged larvae. Additionally, quantitative real-time reverse transcription-PCR analyses demonstrated increased transcriptional rates of immune-related genes at 5 h post-challenge with purified XeGroEL. Our results help to understand anti-microbial immune responses in G. mellonella, suggesting that the immune system recognizes exogenous proteins and pathogen-associated molecular patterns.

Development of immunoassay based on monoclonal antibody reacted with the neonicotinoid insecticides clothianidin and dinotefuran.

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 1.5–15 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.

Long-term effects of malaria prevention with insecticide-treated mosquito nets on morbidity and mortality in African children: randomised controlled trial.

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.

Selection of single-chain variable fragment antibodies against fenitrothion by ribosome display.

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.

Highly sensitive and specific detection of neonicotinoid insecticide imidacloprid in environmental and food samples by a polyclonal antibody-based enzyme-linked immunosorbent assay.

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.