Hapten synthesis, monoclonal antibody generation, and development of competitive immunoassays for the analysis of picoxystrobin in beer

This paper describes the original synthesis of a functionalized derivative of the fungicide picoxystrobin and the generation of the first reported monoclonal antibodies against this strobilurin pesticide. The synthetic hapten was prepared by total synthesis from commercial chemicals and incorporating the spacer arm through a carbon-carbon single bond. Also, to obtain the immunogen, an uncommon hapten activation strategy based on N,N'-disuccinimidyl carbonate was employed, affording high activation yields and clean and reproducible coupling results. With these immunoreagents, two enzyme-linked immunosorbent assays (ELISAs) were developed: a competitive one-step assay using the antibody-coated direct ELISA format and a competitive two-step assay with the conjugate-coated indirect ELISA procedure. Both immunoassays were characterized in terms of sensitivity, selectivity, tolerance to solvents and matrix effects, achieving limits of detection below 0.2 μgL(-1). The optimized assays were used for the determination of picoxystrobin residues in beer, with recovery values ranging between 90 and 121% for the direct assay and from 79 to 122% for the indirect assay.     

Application of internal standard for derivative-spectrophotometric determination of azinphos-methyl in commercial formulations

The use of an internal standard is proposed in this work for first derivative spectrophotometric determination of azinphos in formulations. Generally, the spectrophotometric procedure is simpler and less expensive than chromatographic techniques recommended for the analysis of pesticides. However, while determining the pesticide in commercial formulations the, many-fold dilution required for such sensitive detection is a serious source of analytical error. It is known that an internal standard (IS), if properly chosen, can help eliminate this type of problem. A mixture of acetophenone (used as an IS in the high performance liquid chromatography (HPLC) procedure) and the blue dye Erioglaucine (A) was applied for the determination of azinphos-methyl in commercial formulations. To ensure the best conditions for the zero-crossing technique, the composition of the mixture was optimized to obtain the zero value of the first derivative absorbance of the IS at a minimum of the azinphos-methyl first derivative absorbance. Also, at the maximum of the first derivative spectrum of the IS, the differential absorption signal of the analyte was negligible. Analytical characteristics for the first derivative spectrophotometric procedure proposed were evaluated (r(2) = 0.9998, detection limit = 0.043 mg, quantification limit = 0.143 mg) and the analytical results obtained (35.02 +/- 0.20% of azinphos-methyl in the formulation) were in good agreement with the results obtained using the official HPLC method (35.44 +/- 0.32%).     

Development of an enzyme-linked immuno-sorbent assay (ELISA) method for carbofuran residues

The haptens 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-amino]butanoic acid (BFNB) and 6-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)-carbonylamino]hexanoic acid (BFNH) were synthesized and then used to develop a rapid,specific and sensitive ELISA method to determine residues of the pesticide carbofuran in a variety of matrices. A hybridoma cell line (5D3) producing anti-carbofuran monoclonal antibodies (MAbs) was also established. Based on the MAbs in combination with the heterologous hapten BFNH coupled to either horseradish peroxidase (HRP) or ovalbumin(OVA), four ELISAs (formats I-IV) for the quantification of carbofuran were developed and compared. Among them, the optimized format II (the conjugate-coated direct competitive ELISA) showed the best characteristics, with an IC50 value of 18.49 ng/mL, a limit of detection of 0.11 ng/mL and the shortest assay time (1 h). This ELISA method was then applied to the determinations of carbofuran in environmental water, soil and food samples. The relative standard deviations (R.S.D.s) ranged from 1.8% to 21.3% and the mean recoveries were 104.6%, 108.3%, 106.3% and 100.1% for water, soil, lettuce and cabbage, respectively. Thus, the ELISA method of format II exhibited the potential to develop commercial ELISA kits for a rapid detection of carbofuran for human health and environmental safety.     

Specific polyclonal-based immunoassays for sulfathiazole

A highly sensitive and specific enzyme-linked immunosorbent assay has been developed for detection of sulfathiazole (STZ, 4-amino-N-thiazol-2-yl-benzenesulfonamide). A set of haptens was synthesized in order to produce polyclonal antibodies against sulfonamides. Two ELISA formats (antibody-coated and conjugate-coated) were also investigated using all the serum/coating conjugate combinations that showed specific recognition. The developed ELISA succeeded in detection of STZ at concentrations as low as 0.03 ng mL^–1 over a measurable range of 0.12–6.71 ng mL^–1. Selectivity studies have demonstrated that other sulfonamides do not interfere significantly (<10%) with analysis of STZ by this immunochemical technique. Analysis of spiked bee honey samples by the developed ELISA method showed recoveries were good. The selectivity and sensitivity (IC₅₀=1.6 ng mL^–1) make it a suitable screening method for determination of low levels of STZ in food samples.     

Antibody-invertase Cross-linkage Nanoparticles: A New Signal Tag for Point-of-Care Immunoassay of Alpha-fetoprotein for Hepatocellular Carcinoma with Personal Glucometer

Methods based on immunoassays have been developed for cancer biomarker alpha-fetoprotein (AFP), but most involve complicated or stripping procedures and are unfavourable for routine use. Herein we report the proof-of-concept of simple and point-of-care (POC) immunoassay for AFP in hepatocellular carcinoma on a portable personal glucometer (PGM) by using antibody-invertase cross-linkage nanoparticles as the signal-generation tags. Antibody-invertase cross-linkage nano tags were synthesized by using reverse micellar method with glutaraldehyde. The POC immunoassay was carried out on monoclonal anti-AFP capture antibody-coated microplate with a sandwich-type reaction mode. Introduction of invertase nano labels accompanying target AFP could hydrolyse sucrose into glucose and fructose. The produced glucose molecules could be determined on a portable personal glucometer. Relative to unimolecular invertase labelling, improved analytical features were acquired with antibody-invertase nano labelling. With the nano labels, PGM-based immunoassay exhibited good electrochemical responses for the detection of AFP, and allowed detection of AFP at a concentration as low as 5.4 pg mL⁻¹. Moreover, a good repeatability and intermediate precision could be found down to 12.68 %. Good well-matched results were obtained for analysis of human serum specimens between POC immunoassay and commercial human AFP ELISA kit.     

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.

Analysis of Phosmet and Azinphos-Methyl in Apples by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatography (HPLC) method has been developed to analyze two organophosphate insecticides (phosmet and azinphosmethyl) in apples. The procedure includes a novel extraction whereby whole apples are sonicated for 2 min in 100 ml of MeOH to remove the pesticides. Reversed-phase HPLC separation was accomplished with an Ultremex C18 column and acetonitrile:methanol:water as the eluent. Detection was at 224 nm for phosmet and 300 nm for azinphos-methyl. For both pesticides the limit of detection was 0.5 ppb and the linearity was from 1 to 405 ng injected. Average recoveries were 80% for phosmet and 86% for azinphos-methyl. Thirteen apple varieties comprising 240 apples were analyzed from supermarkets and roadside stands for phosmet (amount found ranged from none detected to 1233 ppb) and azinphos-methyl (amount found ranged from none detected to 388 ppb). Confirmation of phosmet and azinphos-methyl was made by UV spectral scans.     

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.     

Analytical performances of validated chemiluminescent enzyme immunoassays to detect N-methylcarbamate pesticides

In the present work, enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection for the determination of carbofuran, carbaryl and methiocarb were developed and the analytical parameters of these assays were compared with those of ELISAs with colorimetric detection. Both were conjugate-coated formats based on identical monoclonal antibodies and homologous protein conjugates. In comparison with colorimetric ELISA, the ability of the chemiluminescent reagents to detect lower concentrations of horseradish peroxidase allowed to decrease the optimal antibody and conjugate concentrations and to reach better analytical parameters. The experimental comparison of the analytical performance of the ELISAs was carried out by analysing extracts of apple-strawberry baby food and simply diluted fruit juices, both spiked at different concentration levels with the above mentioned pesticides. Recovery values for both ELISAs were around 100% and no matrix effects were observed when fruit juices were diluted 1:20 or more. Results obtained by ELISAs correlated well, both in terms of accuracy and precision, with those obtained by a liquid chromatography-electrospray mass spectrometry (LC/ESI/MS/MS) analysis, used as reference method to validate the immunoassays results. The limits of detection reached by using the chemiluminescent assay were 0.03, 0.007 and 0.004 ng ml⁻¹ for carbofuran, carbaryl and methiocarb, respectively.     

磁敏传感器竞争免疫法检测牛奶中的氯霉素残留量

利用直接竞争免疫原理和巨磁致电阻效应,建立了磁敏生物传感器检测氯霉素的方法。制备氯霉素半抗原芯片,依次加入待测样品、生物素化抗体、链霉亲和素磁颗粒偶联物,使之发生竞争免疫反应,再利用传感器检测芯片上结合的磁颗粒数目。通过对检测条件的优化,建立了氯霉素浓度与磁颗粒数目的标准工作曲线。本方法的检测范围为0.05～100.0μg/L;检出限为50 ng/L;用于牛奶检测,回收率为95.97%～99.36%;批内相对标准偏差为0.8%～3.9%,批间相对标准偏差为1.1%～1.7%;与ELISA方法的一致相关系数达到0.98。本方法可在30 min内快速完成定量检测,为快速多靶标磁敏竞争免疫检测体系的建立提供了可行性。     

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.     

Comparison of Different Elisa Formats for the Detection of Cephalexin and Application Validation

Four ELISA formats, antigen-coated indirect, antigen-coated direct, antibody-coated, and the secondary antibody-coated, were developed using monoclonal antibody to determine cephalexin. Results showed that the secondary antibody-coated method of ELISA had a better performance in the establishment of standard curves. The optimized secondary antibody-coated ELISA was used to determine cephalexin spiked in pig muscle, pig kidney, pig liver, chicken muscle, chicken liver, and cow's milk. The limits of detection were 0.09 ng/g, 0.15 ng/g, 0.26 ng/g, 0.13 ng/g, 0.19 ng/g, and 0.08 ng/mL in pig muscle, pig kidney, pig liver, chicken muscle chicken liver, and cow's milk, respectively. A mean recovery of 77.2–128.5% and coefficient of variation of 2.6–14.7% were obtained. The results given by the ELISA method were in agreement with those of the LC-MS/MS method, which confirmed the potential of the ELISA method for the monitoring of cephalexin in milk and animal tissues.     

A novel competitive fluorescence immunoassay for the determination of dibutyl phthalate

A novel, sensitive, and specific competitive fluorescence immunoassay has been developed for the quantitative determination of dibutyl phthalate (DBP) using an antibody-coated plate format. Hapten was synthesized in order to produce polyclonal antibodies against dibutyl phthalate. Polyclonal antisera to dibutyl phthalate were generated in rabbits and used to construct the fluorescence immunoassay for measurement of dibutylphthalate. The assay had a detection limit of about 0.02 μg L⁻¹, a dynamic range of approximately 0.1–300 μg L⁻¹. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. The study demonstrated that the developed antiserum and fluorescence immunoassay procedure can be used to detect dibutyl phthalate in environmental samples such as tap water, river water, drinking water, and leachate from plastic drinking water bottles.     

Analysis of a mixture of polychlorinated biphenyls and chlorinated pesticides in human serum by column fractionation and dual-column capillary gas chromatography with electron capture detection

An analytical method is presented for precise identification and quantitation of 29 specific polychlorinated biphenyl (PCB) congeners and 15 chlorinated pesticides in human serum. Analyte surrogates PCB 30, PCB 204, 2,2',4,4',5,5'-hexabromo-biphenyl, perthane, alpha-hexachlorocyclohexane, and dichlorobenzophenone were added to each sample. The serum was extracted with an organic solvent and separated by adsorption chromatography into 3 elution fractions for high-resolution gas chromatographic analysis. Each fraction was analyzed by dual-column capillary chromatography followed by electron capture detection. Two capillary columns, DB-5 and DB-1701, with different polarities were used to increase selectivity for each analyte. Quantitation was performed by selecting 2 sets of calibration standard mixtures and 1,2-dichloronaphthalene as an internal standard. Mean recoveries ranged from 39 to 126% for selected analytes and from 31 to 88% for surrogates. Detection limits for specific congeners and pesticides are reported. Typical chromatographic profiles of calibration standard mixtures, as well as a human sample, are illustrated. Verification of each analyte is assessed, and results of analyses of selected human samples and quality control criteria used to ensure data validity also are presented.     

Rapid chemiluminescence biosensing of microcystin-LR

A rapid immunoassay for sensitive detection of microcystin-LR using a portable chemiluminescence multichannel immunosensor (CL-MADAG) was developed. The sensor device is based on a capillary ELISA technique in combination with a miniaturized fluidics system and uses chemiluminescence as the detection principle. Minimum concentrations of at least 0.2 μg L⁻¹ microcystin-LR could be unambiguously measured in a spiked buffer system as well as in spiked real water samples. A single sample analysis for detection of microcystin-LR could be accomplished in just 13 min on the CL-MADAG. Besides providing a highly reproducible, fast and easy to perform test format, one major advantage of the newly established capillary immunoassay is represented by the feasibility of an internal retrospective quality control mechanism. Finally, simultaneous CL-MADAG measurements employing our inhibition immunoassay and a sandwich ELISA could be successfully demonstrated.     

Myeloperoxidase-mediated oxidation of organophosphorus pesticides as a pre-step in their determination by AChE based bioanalytical methods

In order to improve the sensitivity of assays for inhibitors of the enzyme acetylcholine esterase (AChE), an effective method was developed for the conversion of the organophosphate pesticides (OPs) diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate into more toxic inhibitors. This was accomplished by converting them from the thio form into their oxo form using the enzyme myeloperoxidase. The oxo forms, which are the only products of conversion, were determined by AChE bioassays, using either the free enzyme, or a flow injection analysis manifold with immobilized AChE and spectrophotometric detection. All modified OPs exhibited inhibitory power at ppb levels and within 10 min. The method is considered to represent an excellent means for improving the sensitivity of assays for determination of OPs.     

Study of the behaviour of azinphos-methyl in a clay mineral by batch and column leaching

Research efforts dealing with the processes affecting the transport of pesticides in soils are needed in order to prevent further damage of surface and groundwater reserves. Although organic matter has been recognised as the most important contributor to the adsorption of non-ionic organic pesticides in soils, in some cases clay minerals may have an important role in the retention of these compounds. The present study was designed to improve the knowledge of the behaviour of azinphos-methyl in soils. Coefficients from adsorption isotherms and HPLC analysis of soil column leachates were used in this work for predicting pesticide mobility in soils. The studied clay mineral was a Spanish bentonite with a predominant montmorillonite fraction. The results showed that azinphos-methyl was adsorbed on the clay mineral and demonstrated the catalytic effect of bentonite on the hydrolysis of the pesticide.     

An accurate quantitative analysis of polymorphs based on artificial neural networks

Measurement precision based on homogeneous and accurate standard samples has been reported to result in significant improvement in the sensitivity and accuracy of the quantitative analysis of polymorphic mixtures. The purpose of this study was to further improve the accuracy of the quantitation based on data processing by artificial neural networks (ANNs), using such high quality standard samples. Homogeneous powder mixtures of - and γ-forms of indomethacin (IMC) at various ratios (0–50% -form content) were subjected to X-ray powder diffractometry. The two diffraction peaks selected as the best combination in multiple linear regression (MLR) were used in the ANN with an extended Kalman filter as a training algorithm. The results obtained by ANN had better predictive accuracy at lower contents (0–5%) compared to those of MLR. ANNs for the diffraction data based on high quality standard samples provide an extremely precise and accurate quantification for polymorphic mixtures.     

流动注射化学发光免疫分析研究I. 血清中乙型肝炎表面抗原的测定

我们首次以键合有抗体的多孔玻璃作为固相免疫分析的免疫反应器, 以化学发光作为最终检测手段, 建立了一种新的、高效率的免疫分析技术-流动注射化学发光免疫分析技术。实验表明: 采用该技术可使单次测定时间从ELISA(Enzyme-linked Immunosorbent Assay)法的二十多小时降至二十分钟, 且所有操作均可在微机控制下自动完成。用该方法对人血清中乙型肝炎表面抗原的测定结果与ELISA法所得结果一致, 对同一样品连续九次测定的相对标准偏差为7.2%。因此, 该方法具有自动化程度高、分析速度快、稳定性好的优点。     

Determination of pesticides by enzyme immunoassay

Immunochemical methods of analysis, which are based on the binding of an antigen (pesticide) molecule to specific antibodies, are finding increasing use for determining pesticides in various samples (water, soil, food products, and biological fluids). Among these, enzyme-linked immunosorbent assay (ELISA), which combines the unique specificity of immunoassay with the high sensitivity of the detection of an enzymatic marker, is the most widely used method. Moreover, in ELISA, the components of an immunochemical reaction are separated; as a consequence, the effect of interfering components in the sample (a so-called matrix effect) is reduced. In this review, the principles of enzyme immunoassay for pesticides are considered, and the determination of pesticides in environmental samples and food products is exemplified. The main directions of the further development of immunoassay techniques for determining pesticides are also discussed.Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 3, 2005, pp. 230–246.Original Russian Text Copyright © 2005 by Morozova, Levashova, Eremin.