整体柱固相萃取-高效液相色谱法在线分析水果中的氯吡脲

建立了整体柱固相萃取-高效液相色谱/紫外检测在线联用方法并用于水果中氯吡脲的分析测定。考察了萃取溶剂的pH值及其流速、溶剂解吸时间及其流速等因素对氯吡脲萃取率的影响。在优化的条件下,该方法对氯吡脲的富集倍数为214倍,方法的线性范围为0.01~50.00 μg/L,检出限为25 ng/L,相对标准偏差（RSD）为3.9%。黑葡萄、猕猴桃和西瓜等水果中氯吡脲的加标回收率为87.0%~120.7%,RSD为0.6%~9.2%。结果表明该方法快速、灵敏,能满足水果中痕量氯吡脲残留分析的要求。     

Determination of thiabendazole in fruit juices by a new monoclonal enzyme immunoassay

A competitive, indirect enzyme-linked immunosorbent assay (ELISA) for thiabendazole has been developed and applied to the analysis of fruit juices spiked with this fungicide. The immunoassay is based on a new monoclonal antibody derived from a hapten functionalized at the nitrogen atom in the 1-position of the thiabendazole structure. To our knowledge, such a structure has not been previously used to obtain antibodies to thiabendazole. The I50 value and the detection limit of the ELISA for standards were 0.2 and 0.05 ng/mL, respectively. Fruit juices were analyzed by diluting samples in assay buffer, without extraction or cleanup. Samples were not even centrifuged or filtered to remove fruit pulp. Under these conditions, the immunoassay was able to accurately determine thiabendazole down to 1 ng/mL in orange and grapefruit juices, down to 5 ng/mL in banana juice, and down to 20 ng/mL in apple and pear juices. Sensitivity differences of the ELISA were caused by the minimum dilution required by each juice to minimize matrix effects: 1/10 for orange and grapefruit juices, 1/50 for banana juice, and 1/100 for apple and pear juices. In an attempt to further increase the sensitivity of the immunoassay for matrixes showing the strongest interferences, apple and pear juices spiked with thiabendazole at low levels (1-20 ng/mL) were extracted with ethyl acetate before analysis. This simple procedure entailed a significant reduction of matrix effects, which in fact allowed us to determine accurately as low as 5 ng/mL thiabendazole in apple and pear juices. Irrespective of whether samples were analyzed by the direct dilution method or after extraction, the simplicity, sensitivity, and sample throughput of this monoclonal immunoassay makes it a very convenient method for the routine monitoring of thiabendazole residues in fruit juices.     

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.     

液相色谱–串联质谱法测定水果及其制品中氯吡脲残留量

建立了液相色谱–三重四极杆串联质谱测定水果及其制品中氯吡脲的方法。样品经乙腈提取,氨基固相萃取小柱净化后,用ZORBAX Extend-C18柱(150 mm×2.1 mm,5μm)分离,以甲醇–水为流动相等度洗脱,采用多反应监测正离子模式检测,外标法定量。氯吡脲的质量浓度在4.0~200.0 ng/m L范围内线性良好,相关系数大于0.999,在5.0,10.0,20.0μg/kg 3个添加水平下,氯吡脲的平均加标回收率为86%~92%,测定结果的相对标准偏差为5.3%~7.6%(n=5),方法定量下限为2.0μg/kg。方法灵敏度高,操作简便,定量准确,可满足梨、柑桔、黄桃等水果及其罐头制品中氯吡脲残留的检测与确证需要。     

Evaluation of a commercial immunoassay for the detection of chlorfenapyr in agricultural samples by comparison with gas chromatography and mass spectrometric detection

A commercially available enzyme-linked immunosorbent assay (ELISA) kit with a high affinity monoclonal antibody was applied to residual analysis of insecticide chlorfenapyr in agricultural samples, and drawn a parallel between the ELISA and gas chromatography (GC) with mass spectrometry (MS). For standards prepared in water containing 5% (v/v) methanol, the sensitivity (I50 value), the dynamic range, and the limit of detection of the ELISA kit were 2.3, 1 - 10, and 0.1 ng/g, respectively. The used monoclonal antibody in the ELISA kit had a high selectivity. The ELISA kit was applied to the determination of chlorfenapyr in two kinds of fruits (apple and peach). The examination of the influence of these matrices on the reliability of the assay performance indicated that the ELISA could determine it in these samples near the regulation values in Japan simply by diluting the methanolic extract or by concentrating it, without any clean-up procedures. Recovery and precision of the proposed ELISA method were assessed by fortifying fruit samples with chlorfenapyr ranging from 0.05 to 1.5 microg/g. Mean recoveries were 94.2 and 90.3% for apple and peach, and coefficients of variation were below 16% in most cases. The results obtained from the proposed ELISA method correlate well the reference GC/MS method for both fruit samples (r > 0.98). These considerations make the ELISA kit very useful analytical tool for monitoring and regulatory programs, without the need of complex and expensive instrumentation.     

Determination of natural olive fruit fly pheromone in insect samples by enzyme linked immunoassays

The olive fruit fly pheromone avidin-biotin ELISA immunoassay, based on the use of polyclonal G antibodies derived from rabbits (reported previously) and a newer assay, based on the use of polyclonal Y antibodies isolated from the eggs of laying hens (reported in this paper), were applied successfully for the analysis of natural pheromone in virgin adult female olive fruit flies. According to the results obtained, the pheromone content in the glands of adult female olive fruit flies increases from the third to the ninth day of their age. During the calling period, the female olive fruit flies seem to emit approximately 1.1microg pheromone/insect/day at least. The immunoassay, based on the Y antibodies, is slightly more sensitive (detection limit 40ng/mL) than the assay based on polyclonal anti-pheromone rabbit antiserum (detection limit 80ng/mL). As revealed by thorough cross-reactivity studies, including 14 structurally similar to the olive fruit fly pheromone molecules, the newer immunoassay is less selective than the previous one and seems to cross react with few molecules bearing the spiroketal moiety.     

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for sulphamethazine residues in edible animal foods: investigation of the effects of the analytical conditions and the sample matrix on assay performance

To determine sulphamethazine (SMZ) residues in edible animal foods (pig muscle, chicken muscle, egg, fish, milk and liver), a competitive direct enzyme-linked immunosorbent assay (ELISA) and a colloidal gold immunoassay were established. The limits of detection of the ELISA and the colloidal gold immunoassay were 0.02 and 0.5 μg kg⁻¹, respectively. The specificity of the ELISA developed to the SMZ was high according to the results of cross-reactivity testing with 14 kinds of sulphonamides. To obtain a more sensitive immunoassay, buffer solution (30 mmol L⁻¹ phosphate-buffered saline with 0.05% Tween 20, pH 8.5) was optimized through the whole test procedure. A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed, with recoveries between 74 and 117.5%. Matrix effects can be avoided by 1:10 dilution of pig muscle, chicken muscle, egg, fish, milk and liver with optimal buffer. The detection limit of SMZ was 5 μg kg⁻¹ in liver and 2 μg kg⁻¹ in the other five samples. For the validation of the ELISA tests, sample extracts were analysed by ELISA and high-performance liquid chromatography. The results obtained by these two methods showed a good correlation (r ²) which was greater than 0.9. The colloidal gold immunoassay presented in this assay was successfully applied to determine SMZ in pig muscle, milk and fish below or equal to the maximum residue level (20 μg kg⁻¹).     

A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp. toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n = 12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n = 20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n = 18) and the within-assay variation ranged between 4.5 and 9.0%.     

Determination of azadirachtin in agricultural matrixes and commercial formulations by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for azadirachtin (aza), a biopesticide from the neem tree (Azadirachta indica A. Juss). The immunogen was synthesized by epoxidation using the furan ring in the aza molecule. Rabbits were immunized with either bovine serum albumin (BSA)-azadirachtin or ovalbumin (OA)-azadirachtin conjugate. Evaluation of the antisera by antibody capture assay showed that the antibody titer of antisera raised against OA-aza was 1:30,000. An indirect competitive ELISA was developed with BSA-azadirachtin as coating antigen and aza-specific antibodies raised against OA-aza immunogen. The immunoassay showed an inhibitory concentration (IC50) value of 75 ppb, with a range of detection from 0.5 to 1,000 ppb for azadirachtin [based on regression analysis, y= 85.87 (-18.89x); r2 = -0.97]. Cross-reactivity of the antibodies with 2 aza- derivatives (22,23-dihydro-23beta-methoxy azadirachtin and 3-tigloylazadirachtol) was 33 and 29%, respectively. The indirect competitive ELISA was validated and evaluated by quantitating aza in spiked agricultural commodities and from neem formulations. Azadirachtin was spiked into 5 different agricultural commodities: tomato, brinjal, coffee, tea, and cotton seed at 500 and 1,000 ppb and recovered at 62-100%. In samples drawn from 6 lots, the aza content in neem-seed kernels ranged from 0.1 to 0.15%; in commercial neem formulations the content ranged from 200 to 2,000 ppm. The method developed may be applied to environmental monitoring of aza and quality assurance studies of aza-based commercial formulations.     

Analysis of forchlorfenuron and thidiazuron in fruits and vegetables by surface‐enhanced Raman spectroscopy after selective solid‐phase extraction with modified β‐cyclodextrin

β‐Cyclodextrin and its derivatives can selectively bind to various organic molecules in its cavity and provide good applications in sample preparation. Surface‐enhanced Raman spectroscopy is a sensitive technique and has received increasing attention in the last decade. Herein, 3,5‐dimethyl phenyl carbamoylated β‐cyclodextrin bonded silica gel was used as a ssorbent in solid‐phase extraction to selectively enrich forchlorfenuron and thidiazuron followed by determination with surface‐enhanced Raman spectroscopy. It showed excellent selectivity for forchlorfenuron and thidiazuron and the adsorption capacities were 40.0 and 30.0 μg/g, respectively. A rapid and sensitive method based on the modified β‐cyclodextrin solid‐phase extraction coupled with surface‐enhanced Raman spectroscopy was developed. The linear ranges were 30.0–300.0 μg/L for forchlorfenuron and thidiazuron at 1005 and 640 cm^?1, respectively. Both of the limits of detection were 15.0 μg/L, which were significantly lower than the maximum permitted by the National Standard. The recoveries of forchlorfenuron and thidiazuron were 78.9–87.9% for the spiked grape, kiwi, cucumber and tomato, with relative standard deviations of 8.1–13.2%. The results show that this method is sensitive, selective, and relatively time saving, and has great potential in the analysis of trace amounts of plant growth regulators in fruits and vegetables.     

Determination of Ochratoxin A in apples contaminated with Aspergillus ochraceus by using a microfluidic competitive immunosensor with magnetic nanoparticles

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi of the genus Aspergillus and Penicillium that presents carcinogenic, teratogenic and nephrotoxic properties. In this work, we have developed, characterized and applied an immunoassay methodology comprised of magnetic nanoparticles (MNPs) as platform for immobilizing bioactive materials incorporated into a microfluidic system for rapid and sensitive quantification of Ochratoxin A (OTA) in apples (Red Delicious) contaminated with Aspergillus ochraceus. The sensor has the potential for automation and the detection of OTA was carried out using a competitive indirect immunoassay method based on the use of anti-OTA monoclonal antibodies immobilized on 3-aminopropyl-modified MNPs. The total assay time into the microfluidic competitive immunosensor was 16 min, and the calculated detection limit was 0.05 μg kg(-1). Moreover, the intra- and inter-assay coefficients of variation were below 6.5%. The proposed method can be a very promising analytical tool for the determination of OTA in apparently healthy fruits post-harvest and for its application in the agricultural industry.     

Immunodetection of lactosylated proteins as a useful tool to determine heat treatment in milk samples

This paper reports the optimisation of a competitive immunoassay (ELISA) to detect lactosylated proteins in milk samples. The assay employs monoclonal antibodies for lactosylated proteins produced in our laboratory and requires no pre-treatment of the samples other than a dilution step. Monoclonal antibodies were fully characterised in terms of selectivity and cross-reactivity with structurally related molecules and used in a competitive assay format with lactosylated standard proteins (lactosylated ovalbumin). The detection limit for lactosylated ovalbumin was 0.015 microgram ml-1 and the working range was from 0.010 to 40 micrograms ml-1. The data obtained indicate that the ELISA developed is applicable to diluted milk samples and is able to distinguish between milk samples that have undergone different heat treatments (UHT and pasteurised milk).     

Competitive colorimetric triazophos immunoassay employing magnetic microspheres and multi-labeled gold nanoparticles along with enzymatic signal enhancement

The authors have developed a competitive immunoassay for the model pesticide triazophos (TRIAZ). The method is based on the use of only one monoclonal antibody immobilized on multi-labeled gold nanoparticle (AuNP) probes. The immunoassay uses two sets of probe. The first is a multi-labeled AuNP probe that carries monoclonal antibody (mcAb), single-stranded DNA (ssDNA) and horse radish peroxidase (HRP). The second is a magnetic microparticle (MMP) probe that is obtained by coating MMPs with ovalbumin coupled to TRIAZ. Free TRIAZ and MMP-immobilized TRIAZ compete for binding to the mcAb on the surface of the AuNPs. Because TRIAZ is a rather small molecule, it cannot be bound by two antibodies. The competitive immunoassay overcomes the limitations of small molecule detection using one kind of mcAb only. Sensitive transduction of the immunoreaction is accomplished by enzymatically catalyzed amplified. TRIAZ was quantified by a conventional ELISA and by the immunoassay presented here. Both method are highly sensitive and well reproducible. The new assay has a linear response in the 15 ng L^?1 to 40 μg L^?1 TRIAZ concentration range, and a 14 ng L^?1 limit of detection which make it more sensitive than the ELISA. The recovery rate in case of spiked samples ranges from 78.4 to 105%, and the RSD is <20%. A good correlation was further established between the results of the immunoassay and those of GC-MS analysis.

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Graphical abstract Schematic of a competitive colorimetric triazophos immunoassay employing magnetic microspheres (black color) and multi-labeled gold nanoparticles (red color). The assay overcomes the obstacles in pesticide detection and shows higher sensitivity than the conventional ELISA.

     

Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal

A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB₁ was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB₁ were 2.69 and 0.35 ng mL⁻¹, respectively, with a linear range of 0.93–7.73 ng mL⁻¹. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL⁻¹, and the IC₅₀ was 0.89 ± 0.09 ng mL⁻¹ with a linear range of 0.29–2.68 ng mL⁻¹. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB₁ contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems.     

Optimization of a Direct Competitive Enzyme-Linked Immunoassay for Carbofuran and Application to Water Samples

Abstract

A direct competitive enzyme-linked immunosorbent assay (ELISA) for carbofuran was developed, which was based on the anti-BFNB IgG monoclonal antibody (McAb) and a homologous enzyme tracer (BFNB-HRP). The influence of several physicochemical factors (salt, pH, detergent, and solvent) on the immunoassay was studied. For the standard curve, an I₅₀ of 2.98 µg/l and a limit of detection (I₁₀) of 0.27 µg/l was obtained in a high salt concentration buffer (0.08 M PBS, pH 7.0) with 0.03% BSA. A common challenge for immunoassay, time-dependent drift, was effectively circumvented in our study. The optimized ELISA has been used to quantify carbofuran in water samples spiked at different amounts. The excellent recoveries (71% to 130%) achieved confirmed the potential of the immunoassay for monitoring of carbofuran in waters without purification steps.     

A monoclonal antibody-based time-resolved fluoroimmunoassay for chloramphenicol in shrimp and chicken muscle

A time-resolved fluoroimmunoassay (TR-FIA) for determination of chloramphenicol (CAP) in shrimp and chicken muscle was developed. The method was based on a direct competitive immunoassay using europium-labeled anti-CAP monoclonal antibody (MAb) and CAP-ovalbumin as coated antigen. The limit of detection was 0.05 ng g⁻¹ and limit of quantification was 0.1 ng g⁻¹. Recoveries ranged from 101.2 to 112.5% for shrimp and 104.9 to 115.3% for chicken muscle at spiked levels of 0.1-5 ng g⁻¹, with intra-assay and inter-assay variations 8.7-14.6 and 9.6-17.8%, respectively. The results obtained by the TR-FIA and ELISA correlated well. The established TR-FIA was validated for the determination of incurred shrimp samples and confirmed by gas chromatography with microcell electron capture detector (GC-μECD).     

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid,sensitive and reliable immunoassay methods,namely competitive indirect enzyme-linked immunosorbent assay(CI- ELISA)and colloidal gold-based immunochromatographic assay(CGIA),were developed to detect ofloxacin(OFL).The linear range of the CI-ELISAwas from 0.5 to 128 ng/mL with a limit of detection(LOD)of 0.35 ng/mL.Good recoveries were obtained in analyzing simulated swine urine samples.The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min,and test results were read visually without any instrument.     

Hapten design and indirect competitive immunoassay for parathion determination: correlation with molecular modeling and principal component analysis

A novel procedure for parathion hapten design is described. The optimal antigen for parathion was selected after molecular modeling studies of six types of potentially immunizing haptens with the aim to identify the best mimicking target analyte. Heterologous competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed after screening a battery of competitors as coating antigens. The relationship between the heterology degree of the competitor and the resulting immunoassay detectability was investigated according to the electronic similarities of the competitor haptens and the target analyte. Molecular modeling and principal component analysis were performed to understand the electronic distribution and steric parameters of the haptens at their minimum energetic levels. The results suggested that the competitors should have a high heterology to produce assays with good detectability values. An indirect competitive ELISA was finally selected for further investigation. The immunoassay had an IC₅₀ value of 4.79 ng mL⁻¹ and a limit of detection of 0.31 ng mL⁻¹. There was little or no cross-reactivity to similar compounds tested except for the insecticide parathion-methyl, which showed a cross-reactivity of 7.8%.     

Validation of a method for predicting the precision, limit of detection and range of quantitation in competitive ELISA.

The mathematical model for predicting the precision, limit of detection (LOD) and range of quantitation (ROQ) in a competitive enzyme-linked immunosorbent assay (ELISA) proposed by Hayashi et al. (Anal. Chem., 2004, 76, 1295) was validated. The model describes the relative standard deviation (RSD) of concentration estimates by the RSDs of pipetting volumes of analyte, enzyme-conjugated antigen, antibody and substrate solutions, and the standard deviation (SD) of inherent absorbances between the wells in an ELISA plate. For 6 kinds of direct competitive ELISA kits, the LOD and ROQ predicted by the model agreed well with those obtained by experiments with real samples. It was also confirmed that the model is applicable to the prediction of uncertainty that depends on the pipetting error of the viscous antiserum solution. The model was demonstrated to be useful for estimating the LOD and ROQ of competitive ELISA.     

Development and Optimization of an Indirect Enzyme‐Linked Immunosorbent Assay for Thiamphenicol

Abstract

The specificity of an enzyme‐linked immunosorbent assay (ELISA) was modified by changing the chemical structure of the haptenated coating antigen. A competitive indirect immunoassay was developed, evaluated, and validated to measure thiamphenicol (TAP) in eels. The working range of the assay was placed between 0.45 and 8.35 µg/L. Moreover, the influence of several physicochemical parameters, such as incubation time, ionic strength, detergent concentration, and pH, were studied. The specificity of this immunoassay was evaluated using 23 structurally related compounds. Finally, a good correlation was observed between the chromatographic and immunoassay techniques while analyzing five certified eel samples.