Determination of the herbicide acetochlor by fluorescence polarization immunoassay

A fluorescence polarization immunoassay procedure was developed for determining the herbicide acetochlor from the group of chloroacetanilides. Conjugates of fluorescent labeled acetochlor derivatives (tracers) with glycylaminofluorescein and ethylenediaminofluorescein were synthesized. The effect of the tracer structure on the analytical characteristics of the determination and on antigen-antibody binding constants was studied. The developed immunoassay procedures are characterized by a detection limit of 10 ng/mL and an analytical range of 0.01–10 µg/mL. The procedure is highly specific; other pesticides caused no interference with the determination of acetochlor, and the percent cross-reactivities for related chloroacetanilide herbicides were no higher than 2.4% for metolachlor and lower than 0.1% for alachlor.Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 1, 2005, pp. 91–96.Original Russian Text Copyright © 2005 by Deryabina, Yakovleva, Popova, Eremin.     

Trace analysis of pollutants by use of honeybees, immunoassays, and chemiluminescence detection

Specific and sensitive analysis to reveal and monitor the wide variety of chemical contaminants polluting all environment compartments, feed, and food is urgently required because of the increasing attention devoted to the environment and health protection. Our research group has been involved in monitoring the presence and distribution of agrochemicals by monitoring beehives distributed throughout the area studied. Honeybees have been used both as biosensors, because the pesticides affect their viability, and as “contaminant collectors” for all environmental pollutants. We focused our research on the development of analytical procedures able to reveal and quantify pesticides in different samples but with a special attention to the complex honeybee matrix. Specific extraction and purification procedures have been developed and some are still under optimization. The analytes of interest were determined by gas or liquid chromatographic methods and by compound-specific or group-specific immunoassays in the ELISA format, the analytical performance of which was improved by introducing luminescence detection. The range of chemiluminescent immunoassays developed was extended to include the determination of completely different pollutants, for example explosives, volatile organic compounds (including benzene, toluene, ethylbenzene, xylenes), and components of plastics, for example bisphenol A. An easier and portable format, a lateral flow immunoassay (LFIA) was added to the ELISA format to increase application flexibility in these assays. Aspects of the novelty, the specific characteristics, the analytical performance, and possible future development of the different chromatographic and immunological methods are described and discussed.

Fluorescence polarization immunoassays for pesticides

Fluorescence polarization immunoassay methods for the detection of pesticides and their metabolites or degradation products are reviewed. Advantages and limitations for application to pesticide detection in environmental and food samples are discussed. The influence of the structure of fluorescent-labeled tracers and the affinity and specificity of antibodies on analytical performance is examined. The methods are simple, readily automated, and rapid (total time for assay of a water sample is about 1 min) with sensitivity of 1 - 10 ng/ml pesticide in 0.01 - 0.1 ml sample.     

Functionalized polymeric magnetic nanoconstructs for selective capturing and sensitive detection of Salmonella typhimurium

Rapid detection and enumeration of pathogens is essential for monitoring contamination and spoilage of food products to ensure improved quality control management. Functionalized polymeric magnetic nanoconstructs (FPMNCs) were developed as an effective immunomagnetic separator and sensing platform for the selective capturing of Salmonella typhimurium. Novel FPMNCs were prepared in three stages involving synthesis of iron oxide (IO) dispersion, capping with sodium oleate and encapsulation of preformed IO nanoparticles by in-situ free radical emulsion polymerization of styrene (St), methyl methacrylate (MMA) and acetoacetoxy ethylmethacrylate (AAEM). PMMA improves the stability of FPMNCs by bridging extremely hydrophobic PS and hydrophilic PAAEM. Core-shell morphology of hydrophobic core of IO, PS & PMMA and hydrophilic shell of PAAEM was demonstrated by SEM, TEM and FTIR studies. FPMNCs with surface functionalized acetoacetoxy groups were covalently attached with polyclonal antibodies against Salmonella common structural antigen (CSA-1-Ab) without using any linker and catalyst. Colorimetric readout signal was acquired using CSA-1-Ab-HRP as secondary antibody after formation of sandwich immunocomplex with bacteria where the optical density of the samples were recorded using ELISA plate reader at 450 nm. The developed immunoassay was specific and selective which captures only targeted S. typhimurium with a detection limit of 10 cells/mL lower than infectious dose of salmonellosis infection. Minimal interference of food matrix with high signal to noise ratio was shown by various food samples. In addition, the performance of developed FPMNC based immunoassay was superior to commercially available immunomagnetic microbeads demonstrating undisputed advantage for capturing and detecting specific bacteria without any pre-enrichment of sample.     

Immunochemical determination of four organophosphorus insecticide residues in olive oil using a rapid extraction process

Sensitive, simple and rapid ELISA methods have been developed for the determination of four organophosphorus pesticides in extra virgin olive oil. The analytical procedure involves simultaneous extraction of the analytes from oil matrix with methanol and a freezing clean-up step (−80 °C), followed by immunoassay determination using standards in matrix. The methodology is specific for diazinon, fenthion, malathion and chlorpyrifos showing little or no cross-reactivity against other organophosphorus compounds. Limits of detection for the pesticides in olive oil are from 46 ng ml⁻¹ for diazinon to 10 ng ml⁻¹ for fenthion, all of them under the established MRLs for olives. The excellent recoveries (between 94 and 122%) obtained by the complete analytical protocol confirm the potential of this approach for detecting these compounds in olive oil, being useful as screening and complementary method in pesticide regulatory and food safety programs. The proposed methodology also correlates well with the reference chromatographic (GC-MS) methods.     

Analysis of Biogenic Amines Using Immunoassays,HPLC, and a Newly Developed IC-MS/MS Technique in Fish Products—A Comparative Study

In this study, comparative analyses were carried out with ion chromatography mass-spectrometry (IC-MS/MS) which has no derivatization step, high-performance liquid chromatography (HPLC) technique, as well as two quantitative and two semi-quantitative immunoassays. The results demonstrated that HPLC and quantitative immunoassay methods were well-correlated with IC-MS/MS in determining histamine in various types of fish products. The best correlation was observed with the HistaSure ELISA Fast Track kit (R² = 0.9903). More than half of the values (68%) obtained by two methods were also statistically similar. The results of semi-quantitative test kits also supported histamine values estimated by quantitative methods, with some exceptions. The best results were found for HistaSure Lateral Flow in supporting the quantitative techniques. Therefore, these methods are found suitable for monitoring histamine in fish products in terms of food safety. Good correlations were also observed HPLC and IC-MS/MS in determining cadaverine, putrescine, and tyramine with the highest value observed for tyramine as R² = 0.9785. However, no correlation was observed for other biogenic amines, and the majority of the results were significantly different from each other for these amines (p < 0.05). The differences may be caused by the drawbacks reported previously for HPLC. However, further studies are required to confirm the possible effects. This study provides a comparative evaluation of several methods in terms of their suitability in determining biogenic amines in fish products for both monitoring and regulatory purposes.     

The αPROX assay: fluorescence screening of the inhibitory effects of hydrophilic antioxidants on protein oxidation

We report on a new and convenient high-throughput fluorescence technique for determining antioxidant capacities of hydrophilic food samples. The new method is called αPROX (anti protein oxidation) and is based on an equimolar complex of diphenylhexatriene propionic acid (DPHPA) and bovine serum albumin (BSA) in aqueous buffer at pH 7.4. DPHPA is a reporter fluorophore that becomes nonfluorescent upon free radical-induced oxidation. In a typical assay, the DPHPA/BSA complex is challenged with peroxyl radicals and shows almost the same susceptibility to oxidation as unlabeled BSA. The progress of protein oxidation and its inhibition by antioxidants at physiological pH is determined from the time-dependent decrease in DPHPA fluorescence intensity. The αPROX method was compared to other techniques frequently used to measure antioxidant capacities. In this article, representative results are provided for the inhibitory effects of pure food components, fruit juices, wines, and various polar plant extracts on protein oxidation.      

Biosensor immunoassay for the screening of dioxin-like polychlorinated biphenyls in retail fish

Dioxin-like polychlorinated biphenyls (DL-PCBs) often make up the majority of the toxic equivalent (TEQ) contribution of dioxins found in fish samples. For the purpose of making risk assessments, it is therefore important to develop screening methods for determining TEQ concentrations of DL-PCBs in retail fish. We have developed a rapid biosensor immunoassay (BIA) for DL-PCBs that uses a surface plasmon resonance sensor (Biacore 3000). The BIA is highly specific for 2,3',4,4',5-pentachlorobiphenyl (PCB 118) that is generally the most abundant DL-PCB isomer found in fish. The fish extracts were first cleaned up on a multilayer silica gel column followed by an alumina column, then subjected to the assay. The quantitative limit of the assay was 1 ng PCB 118 per gram of tested sample. Dilution and recovery tests using purified fish extracts suggested that the matrix effect was minimized in the assay by diluting the analyzed samples. The assay results for retail fish samples (n=7) agreed well with those obtained by an enzyme-linked immunoassay (ELISA) using the same monoclonal antibody: ELISA has been already validated for determining DL-PCBs in fish samples, so BIA performs well in this analysis. Finally, BIA results for the TEQ concentrations of DL-PCBs in retail fish samples (n=10) correlated well with those obtained by high-resolution gas chromatography coupled to high-resolution mass spectrometry (r=0.89). Our method is therefore useful for screening retail fish to determine the TEQ concentrations of DL-PCBs.     

Chemiluminescent Detection of Gatifloxacin Residue in Milk

Abstract

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detection of gatifloxacin residue in milk was developed in this study. Compared with conventional colorimetric ELISA using the same antibody, the developed CL immunoassay shows a significant improvement in sensitivity and detectability with an IC₅₀ of 0.4 ng mL^?1 and a detection limit of 0.001 ng mL^?1 and thus is suitable to be used as a highly sensitive screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to detect milk samples spiked by gatifloxacin, and satisfactory results were obtained.     

Analysis of erythritol in foods by polyclonal antibody-based indirect competitive ELISA

Sugar alcohols are widely used as food additives and drug excipients. Erythritol (INS 968) is an important four-carbon sugar alcohol in the food industry. Erythritol occurs naturally in certain fruits, vegetables, and fermented foods. Currently, HPLC and GC methods are in use for the quantification of erythritol in natural/processed foods. However, an immunoassay for erythritol has not been developed so far. We have utilized affinity-purified erythritol-specific antibodies generated earlier [9] to develop an indirect competitive ELISA. With erythritol–BSA conjugate (54 mol/mol; 100 ng/well) as the coating antigen, a calibration curve was prepared using known amounts of standard meso-erythritol (0.1–100,000 ng) in the immunoassay. Watermelon (Citrullus lanatus) and red wine were selected as the food sources containing meso-erythritol. The amount of meso-erythritol was calculated as 2.36 mg/100 g fresh weight of watermelon and 206.7 mg/L of red wine. The results obtained from the immunoassay are in close agreement with the reported values analyzed by HPLC and GC (22–24 mg/kg in watermelon and 130–300 mg/L in red wine). The recovery analyses showed that added amounts of meso-erythritol were recovered fairly accurately with recoveries of 86–105% (watermelon) and 85–93.3% (red wine). The method described here for erythritol is the first report of an immunoassay for a sugar alcohol. Figure Indirect competitive ELISA for quantitation of erythritol     

Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: An anticipated analytical tool for food safety

In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator.Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 μg L⁻¹ and EC₅₀ 0.079 μg L⁻¹ were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.     

Performance of two monoclonal immunoassays in mixtures of cross-reacting dithiophosphorus pesticides

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.     

New Developments in Crystallization Processing

The intention of this work presented is to introduce new processing principles for the crystallization of watery or fatty phases in food systems, and in particular to quantify the process — microstructure — product quality relationships for such food systems. The crystallization processes demonstrated in detail are high shear crystallization (i), spray crystallization (ii) and spray powder based seed crystallization (iii). Crystalline, semi-crystalline and/or amorphous structures were analysed by calorimetric, mechanical/rheological and microscopical methods. Quality aspects of the final food products, which are related to the structure of the crystalline and/or amorphous components, were investigated additionally.This revised version was published online in November 2005 with corrections to the Cover Date.     

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance

A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA–BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL⁻¹, and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74–110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL⁻¹ for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R ² = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples.     

Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 μg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n?=?146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 μg/kg by ELISA which correlated to >10 μg/kg by LC-MS/MS.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within ±20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30 ng/mL by ELISA and 0.2 ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10 mL of urine converted into 1 mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.     

Determination of pesticide residues by GC-MS using analyte protectants to counteract the matrix effect.

An analytical method was developed to determine pesticides of various chemical classes in soil, juice and honey using analyte protectants to counteract the enhancement of the chromatographic response produced by the presence of matrix components (matrix effect). This effect was more pronounced for soil and honey samples than for juice samples; regarding the pesticide chemical class, organochlorine pesticides were less affected by the presence of matrix components than triazines and organophosphorus pesticides. Several analyte protectants (2,3-butanediol, L-gulonic acid gamma-lactone, corn oil and olive oil) were tested for counteracting the observed matrix effect. L-Gulonic acid gamma-lactone was an effective protecting agent for most of the pesticides studied in soil and honey samples, whereas olive oil was very effective for juice samples. The combination of these two protectants was found to be an effective analyte protectant for all compounds in soil and honey samples.     

A sensitive immunoassay based on direct hapten coated format and biotin-streptavidin system for the detection of chloramphenicol

A novel enzyme liked immunosorbent assay (ELISA) was developed for the detection of chloramphenicol (CAP). In this assay, the small molecular hapten (Hap) was directly coated on the surface of microtiter plates and biotin-streptavidin system (BSAS) was employed to improve the sensitivity of immunoassay (BSAS-direct Hap coated ELISA). The surface of microtiter plates was treated with glutaraldehyde (GA) polymer network to introduce aldehyde group, which was used to cross-link with amino group of CAP. Compared with conventional ELISA (the plates were coated with Hap-carrier protein conjugates), the modified plates presented significantly high antibody and antigen (Ab-Ag) affinity and showed excellent stability. And then the biotinylated monoclonal antibody (mAb) and HRP-labeled streptavidin were employed in this assay for amplification of signals. The sensitivity of BSAS-direct Hap coated ELISA was increased by approximately 20-folds and the stability was also improved greatly compared to conventional ELISA. Its 50% inhibition concentration (IC₅₀) for CAP was 10.5 ng mL⁻¹ and the limit of detection (LOD) was 0.2 ng mL⁻¹ after optimization of reaction conditions. To our knowledge, this was one of the most sensitive immunoassay for CAP yet reported. In sample analysis, the results of CAP detected by this assay were in accordance with which obtained by conventional ELISA and high performance liquid chromatography (HPLC). Therefore, it is an attractive alternative compared to conventional immunoassays in routine supervision for residue detection in food and environment.     

Pesticides: Behavior in Agricultural Soil and Plants

This review considers potential approaches to solve an important problem concerning the impact of applied pesticides of various classes on living organisms, mainly agricultural crops used as food. We used the method of multi-residual determination of several pesticides in agricultural food products with its practical application for estimating pesticides in real products and in model experiments. The distribution of the pesticide between the components of the soil-plant system was studied with a pesticide of the sulfonylureas class, i.e., rimsulfuron. Autoradiography showed that rimsulfuron inhibits the development of plants considered as weeds. Cereals are less susceptible to the effects of pesticides such as acetamiprid, flumetsulam and florasulam, while the development of legume shoots was inhibited with subsequent plant death.     

Comparative study of three immunoassays based on monoclonal antibodies for detection of the pesticide parathion-methyl in real samples

Three immunoassay systems: indirect, direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA), fluorescence polarization immunoassay (FPIA) based on monoclonal antibodies for the detection of parathion-methyl (PM) were developed and optimized. Several PM derivatives (haptens) were conjugated to proteins and fluoresceinthiocarbamyl ethylenediamine (EDF) to obtain immunogens and competitors. The influence of immunogen and competitor structures on the assay performance was investigated. IC-ELISA was the most sensitive of all techniques developed, with a detection limit of 0.08 ng ml⁻¹, but assay time was the longest (3.5 h per 96-well microtitre plate). DC-ELISA was easier to perform and quicker (1.5 h per 96-well microtitre plate) but less sensitive than IC-ELISA (detection limit was 0.5 ng ml⁻¹). FPIA was the fastest and simplest (7 min per 10 samples) but the least sensitive (detection limit was 15 ng ml⁻¹) technique. The methods were characterized by high specificity and reproducibility. The cross-reactivity for parathion-ethyl was around 30-40% for IC-ELISA and FPIA, but significantly higher (125%) for DC-ELISA. The immunoassays were applied to the analysis of PM residues in different food and environmental matrices. Methanol extracts of vegetable, fruit and soil samples were used for the analysis. Recoveries for most spiked samples averaged between 85 and 110%. The methods developed can be used for screening of food and environmental samples for PM residues without complicated clean-up.