Rapid, on-site and quantitative paper-based immunoassay platform for concurrent determination of pesticide residues and mycotoxins.

Mycotoxins and pesticides are prevalent in cereal food. It is difficult to detect these two kinds of hazard factors simultaneously in rapid assay. In order to find a solution to the problem, carbamates and aflatoxins were selected in this study to establish a rapid, on-site, and quantitative paper sensor. Two novel monoclonal antibodies (mAbs) against carbaryl and carbofuran (1D2 and G11) were developed. The IC50 values (half maximal inhibitory concentration) were 0.8 ng/mL and 217.6 ng/mL for carbaryl and carbofuran, respectively. Based on the sensitive and specific mAbs, a multi-TRFICA (time-resolved fluorescence) paper sensor was developed, which simultaneously detected six types of hazardous chemicals, including AFB1, AFB2, AFG1, AFG2, carbaryl, and carbofuran. A universal sample pretreatment method for mycotoxins and pesticides was explored to apply on established competitive indirect enzyme-linked immunosorbent assay (icELISA) and multi-TRFICA-paper sensor. The established paper sensor can be easily observed with naked eyes, qualitatively under a UV lamp, and quantitated using a home-made device. It exhibited a calculated limit of quantity for AFTs, carbaryl, and carbofuran of 0.03, 0.02, and 60.2 ng/mL in corn samples, respectively. The spiking-recoveries and real sample studies proved that multi-TRFICA-paper sensor is an accurate, sensitive, and high throughput detection method for simple and low-cost analysis in corn samples.

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.

Love wave immunosensor for the detection of carbaryl pesticide.

A Love Wave (LW) immunosensor was developed for the detection of carbaryl pesticide. The experimental setup consisted on: a compact electronic characterization circuit based on phase and amplitude detection at constant frequency; an automated flow injection system; a thermal control unit; a custom-made flow-through cell; and Quartz /SiO2 LW sensors with a 40 µm wavelength and 120 MHz center frequency. The carbaryl detection was based on a competitive immunoassay format using LIB-CNH45 monoclonal antibody (MAb). Bovine Serum Albumin-CNH (BSA-CNH) carbaryl hapten-conjugate was covalently immobilized, via mercaptohexadecanoic acid self-assembled monolayer (SAM), onto the gold sensing area of the LW sensors. This immobilization allowed the reusability of the sensor for at least 70 assays without significant signal losses. The LW immunosensor showed a limit of detection (LOD) of 0.09 µg/L, a sensitivity of 0.31 µg/L and a linear working range of 0.14-1.63 µg/L. In comparison to other carbaryl immunosensors, the LW immunosensor achieved a high sensitivity and a low LOD. These features turn the LW immunosensor into a promising tool for applications that demand a high resolution, such as for the detection of pesticides in drinking water at European regulatory levels.

Development of enzyme linked immunoassay for the simultaneous detection of carbaryl and metolcarb in different agricultural products.

A direct competitive enzyme linked immunosorbent assay in multi-enzyme tracers format for the simultaneous analysis of carbaryl and metolcarb in agricultural products is described in this study. The concentrations of coating antibodies and enzyme tracer were studied. Under the optimum conditions, the limits of detection of carbaryl and metolcarb were 0.15 microg L(-1) and 1.2 microg L(-1), respectively. Determination of carbaryl and metolcarb in fruit juices and vegetables was accomplished by simple, rapid and efficient extraction methods. Recoveries of spiked samples were great than 70%. Validation of the immunosorbent assay was conducted by comparison of results from high performance liquid chromatography (HPLC). The correlations between the data obtained using multi-enzyme tracers enzyme linked immunosorbent assay and high performance liquid chromatography were good. Results indicated that the new strategy for developing immunoassay for simultaneous quantitative determination of carbaryl and metolcarb residues was suitable in this study.

Catalytic antibody therapy against the insecticide carbaryl.

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.

Monoclonal enzyme immunoassay for the analysis of carbaryl in fruits and vegetables without sample cleanup.

The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of 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 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl 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 carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, 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 = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput.

Effects of combinations of endosulfan, dimethoate and carbaryl on immune and hematological parameters of rats.

IgG and IgM levels and hematological parameters (red and white blood cell counts, thrombocyte, monocyte, lymphocyte and granulocyte counts and hemoglobin concentrations) were determined in albino rats exposed to combinations of endosulfan, dimethoate and carbaryl. Two and 3 combinations of 100- and 1000-fold acceptable daily intake (ADI) of endosulfan (ADI = 0.00612 mg/kg), dimethoate (ADI = 0.0204 mg/kg) and carbaryl (ADI = 0.0101 mg/kg) were administered po to male albino rats for 3.5 mo. Animals were immunized s.c. with tetanus toxoid in Freund's complete adjuvant 20 d before terminating exposures. At 100-fold ADI dosing, administration of each of the pesticides alone did not cause any difference in the parameters, but numbers of white blood cells and monocytes increased in rats given endosulfan + dimethoate while numbers of red blood cells increased with dimethoate + carbaryl. Rats given 1000-fold ADI endosulfan + dimethoate + carbaryl had significant differences in almost every parameter, while IgG, IgM white blood cells and lymphocytes decreased, and monocytes and % granulocytes increased from single endosulfan dosing. Lymphocyte counts were reduced by single dimethoate or carbaryl dosing. Endosulfan + dimethoate + carbaryl produced the most effective changes in comparison to single dosing or other pesticide combinations.

Selection and characterisation of membranes by means of an immunofiltration assay. Application to the rapid and sensitive determination of the insecticide carbaryl.

The characterisation and selection of membranes by means of an immunofiltration assay is described. The chemical composition of the membranes was: nitro-cellulose, polyamide, polyvinylidene difluoride, polyethersulfone, cellulose acetate, regenerated cellulose, cellulose nitrate, and glass fibre. In order to characterise the membranes according to their binding capacity, immobilisation stability, sensitivity and hydrodynamic properties, two basic immunofiltration formats were performed. In both formats, enzyme label (horseradish peroxidase, HRP) and colorimetric detection were used. In the immobilised antibody format, three monoclonal antibodies (mAb) against the insecticide carbaryl were immobilised on the membranes by passive adsorption. In the immobilised hapten format, two haptens conjugated to bovine serum albumin (BSA) were immobilised. Immobilon-P was the best membrane with regard to the characterisation criteria and permitted the filtration of large volume (5.0 ml) through the membrane without release of the receptor. The immobilisation of the receptor (antibody or haptenic conjugate) was pH dependent. Good results with regard to mAb-antigen recognition, were obtained using 50 mM carbonate/bicarbonate buffer, pH 9.6. However, the most sensitive assays were achieved using, 10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCI (PBS), pH 7.4 as immobilisation buffer. Furthermore, all these results permit the choice of the best membrane for the rapid and sensitive determination of carbaryl. This study will assist the development of dipsticks, immunoelectrodes, membrane-based immunoreactors or immunoconcentration devices that are based on the use of membranes as immunosupports.

Immunofiltration: a methodology for preconcentration and determination of organic pollutants.

A new concentration procedure using an immunofiltration-based method is described. The approach enables quantitative determination of organic pollutants by filtering large volumes of sample through a poly(vinylidene difluoride) membrane where antibodies have been immobilized by passive adsorption. The analysis is based on a sequential competitive enzyme immunoassay. A wide range of sample volumes have been tested (0.2-5.0 mL) for each type of antibody. The improvement on the assay sensitivity and specificity achieved by means of this concentration procedure is discussed. Using this technique and the insecticide carbaryl as a model analyte, a concentration factor of at least 13 and a limit of detection of 4.75 ng/L are accomplished. The suitability of this methodology is demonstrated by the quantification of the insecticide in several types of water samples (bottled, estuarine, and physiological-saline solutions) with recoveries ranging between 102 and 111%. This method has proved to concentrate carbaryl directly, in an accurate way, for residue analysis without using organic solvents or any extraction process. Furthermore, this procedure offers the advantages of carrying out in the same system both preconcentration and quantitative determination of the analyte.

A comparative study by the enzyme-linked immunofiltration assay of solid phases used in the development of flow immunosensors.

The application of an inert membrane-based, enzyme-linked immunofiltration assay (ELIFA) to the characterization of immunosorbents suitable for flow immunosensor development is described. For direct assays, eight monoclonal antibodies (MAb) raised against the insecticide carbaryl were immobilized on three sorbents, namely, controlled pore glass (CPG), hydrazide derivatized agarose beads and a hydrophilic polymer with immobilized Protein A/G. The interaction between immobilized antibodies and antigen was directly detected using a carbaryl hapten conjugated to horseradish peroxidase. Immunosorbent characterization was based on both sensitivity and re-usability. Optimal immunosorbent regeneration was achieved using 0.1 M glycine/HCl, pH 2.0 as the desorbent solution. The best covalent immunosorbent was obtained by immobilizing LIB-CNA36 MAb on hydrazide derivatized agarose beads. The best immunosorbent obtained by reversible immobilization was LIB-CNH45 MAb on Protein A/G. Using this support the eventual irreversible denaturation of covalently immobilized MAbs was overcome. For indirect assays, N-hydroxisuccinimide derivatized agarose beads and glutaraldehyde-activated CPG were used as sorbents for hapten immobilization via the amino groups of a carrier protein. In this format, antigen-MAb interactions were detected using a peroxidase-conjugated rabbit anti-mouse immunoglobulin. The highest sensitivity was achieved by LIB-CNH45 MAb in combination with derivatized agarose beads. All these results demonstrated the suitability of ELIFA as a fast, precise and easy-to-use technique for immunosorbent selection.

[Effect of a combination of 3 pesticides on the sensitization of guinea pigs to ovalbumin]. / Vliianie na kombinatsiia ot tri pestitsida vurkhu sensibilizatsiiata na morski svincheta s ovalbumin.

The author treated guinea pigs with a combination of three pesticides: First group -- with cyneb in a dose of 12,5 mg/kg of body weight (threshold value) +/- sevine in a dose of Ip 5 mg/kg (twice threshold value) +/- tribufon in a dose of 5.0 mg/kg (threshold value); second group -- cyneb 125 mg/kg (ten times of threshold value) +/- sevine 1.5 mg/kg (twice the threshold value) +/- tribufon 5.0 mg/kg (threshold value). Two groups of animals were used as controls: first group-- treated with 2,0 mg/kg of cyclophosphamide and second group -- with 3% of starch emulsion, with which other substances were given. The indicated substances were administered in the animals by a probe (a catheter) daily, in the morning before feeding, for a period of six months. Then sensibilization was made by ovalbumin in a solution of 10 gm% according the scheme: first injection of 2 ml of ovalbumin -- 1 ml subcutaneously in the inguinal region and 1 ml intraperitonealy. A challenging dose of 2 ml was administered inraperitonealy after 20 days and the reaction of the experimental animals was examined for a period of 90 minutes after the administration of the challenging dose. There was no difference in the reaction of the guinea pigs, treated with a combination of three pesticides in comparison with animals, given the same pesticides in the same dosage, but singly.