Development of competitive enzyme-linked immunosorbent assays for boscalid determination in fruit juices

Boscalid is a modern, broad-spectrum carboxamide pesticide highly efficient against most fungal diseases affecting valuable crops. In this study, a boscalid-mimicking derivative with a six-carbon spacer arm replacing the chlorine atom at the pyridine ring of the target molecule was synthesized and coupled to carrier proteins. Following rabbit immunization, antibodies against this agrochemical were obtained for the first time, and they were characterised in terms of affinity and specificity, tolerance to solvents, and robustness to changes in buffer pH and ionic strength, using two assay formats. Both of the optimised immunoassays showed limits of detection below 0.1 μg/L. Moreover, matrix effects of grape, peach, apple, and tomato juices were evaluated. Finally, a simple and easy procedure was set up for boscalid determination with spiked samples, affording limits of quantification of 10 μg/L, a value well below the sensitivity levels required for monitoring campaigns of pesticide residue analysis in food.     

A monoclonal antibody-based sensitive enzyme-linked immunosorbent assay (ELISA) for the analysis of the organophosphorous pesticides chlorpyrifos-methyl in real samples

Chlorpyrifos-methyl hapten, O-methyl-O-(3,5,6-trichloro-2-pyridinyl)-N-(2-carboxyethyl)-phosphoramidothionte (H1), was synthesized and conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) by the active ester method. Then H1–OVA conjugate was used as coating antigen, while H1–BSA conjugate was used as immunogen for producing monoclonal antibody. After optimisation, a monoclonal antibody-based effective competitive indirect enzyme-linked immunsorbent assay (ELISA) was developed and applied for determination of chlorpyrifos-methyl with a novel combination of antibody/antigen, I₅₀ of which was 75.22 ng/ml, limit detection (LD) was 0.32 ng/ml, and there was relative high cross-reactivity (CR) only with chlorpyrifos (1.4%), and CRs with other tested pesticides were all below 1% and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to real samples, including grape, Chinese cabbages, water and soil were all from 82.4% to 110.2%. Therefore, the optimised ELISA might become a convenient and satisfied analytical tool for monitoring chlorpyrifos-methyl residues in agriculture ecosystem.     

Development of an enzyme-linked immunosorbent assay for diniconazole in agricultural samples

Diniconazole hapten was synthesized and conjugated to bovine serum albumin (BSA) by the carbodiimide method to produce an immunogen and to ovalbumin (OVA) by mixed anhydride method to produce a coating antigen. Polyclonal antibody against diniconazole was generated by immunizing New Zealand rabbits with the immunogen. Under optimised conditions (20% methanol, 0.4 mol/L Na⁺, pH 7.5), an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for detecting diniconazole. The 50% inhibitory concentration (IC₅₀) was 0.071 ± 0.013 mg/L and the limit of detection (LOD) was 1.28 ± 0.80 μg/L. Triazole fungicide analogues of diniconazole were tested and did not obviously cross-react, except for uniconazole (2.25%). The recoveries obtained after addition of standard diniconazole to agricultural samples, including water, soil, pear, grape, tomato and wheat flour, ranged from 70% to 120%. The ic-ELISA developed could successfully be applied to analysis of diniconazole residues in agriculture samples.     

呋喃西林代谢物多克隆抗体制备及酶联免疫吸附分析方法

为了建立检测呋喃西林代谢物(SEM)的酶联免疫吸附分析方法(ELISA),将所设计合成的系列半抗原与牛血清白蛋白(BSA)偶联制备免疫原并免疫新西兰大白兔,筛选获得源于新颖半抗原H3的具有高亲和力、高特异性抗SEM多克隆抗体。同时,基于设计合成的系列同/异源包被抗原,考察了不同结构包被原对ELISA灵敏度的影响,发现H4-OVA作为异源包被原建立SEM的ELISA检测方法可获得最佳的检测效果,结果显示:ELISA方法的半抑制浓度(IC50)为12.37ng/mL;定量检测线性范围(IC20～IC80)为0.439～110.78ng/mL;检测限(IC10)达0.07ng/mL,达到了国内外相关检测限量要求,可应用于实际食品样品检测。     

间接竞争酶联免疫法检测水样中的重金属镉

利用重金属镉多克隆抗体,建立一种低仪器成本的检测水样中重金属镉的间接竞争酶联免疫法,其检测限(IC15)为0.76μg/L,灵敏度(IC50)为11.33μg/L。交叉反应结果表明,该抗体除与汞螯合物的交叉反应率为10.9%,与其他金属(锰,铬,镁,铁,铅,镍,银和铜)螯合物的交叉反应率均低于1.32%。自来水和河水样品中镉的添加回收率为93.95%～107.40%,变异系数为3.97%～14.69%。     

Immunological detection of moulds in food by using the enzyme-linked immunosorbent assay (ELISA); preparation of antigens

Antigens were isolated from moulds (Penicillium verrucosum var. cyclopium, Mucor racemosus and Fusarium oxysporum) by a simple water extraction followed by column chromatography using Sepharose CL4B. The antigens obtained were genus-specific, heat-stable and found not to be present in extracts of non-moulded food. Immunoglobulins were produced against these antigens and an enzyme-linked immunosorbent assay (ELISA) was developed. Using the ELISA, moulds were detected in both heated and unheated foods in a sensitive and specific way. The results indicate the possibility of testing whether prepared foods are produced from non-moulded raw materials or not. Heat treatment during processing does not influence the recovery of mould contamination.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay for determination of doxycycline in chicken muscle,liver and egg

A modified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed using a highly sensitive and specific monoclonal antibody (McAb) to determine doxycycline (DC) residues in chicken tissues and egg. The McAb against DC was produced by hybridoma technique and a modified ic-ELISA was characterised in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed at concentrations ranged from 0.01 to 100 ng/ml. The IC₅₀ value was 1.32 ± 0.18 ng/ml. The limit of detection was 0.14 ± 0.02 ng/g. The recoveries of DC from spiked chicken liver, muscle, and egg at levels of 50–600 ng/g were 84.6–85.5%, 88.2–89.1%, and 84.4–89.3%, respectively. The coefficient variations (CVs) were 5.1–9.3%, 3.7–11.3%, and 4.7–9.8%, respectively. Linear regression analysis showed good correlation, with r² values 0.9909 for chicken liver and 0.9916 for chicken muscle.     

直接竞争ELISA法快速测定水产品中6种氟喹诺酮类药物

采用过碘酸钠法将抗环丙沙星单克隆抗体（MAb-CIP）与辣根过氧化物酶（HRP）偶联制备酶标抗体MAbCIP-HRP,建立了检测水产品中6种氟喹诺酮类药物残留的直接竞争酶联免疫吸附分析方法（dcELISA）,考察了包被原浓度、竞争反应时间和有机溶剂等因素对方法灵敏度的影响。结果表明:在优化的反应条件下,所建立的dcELISA针对环丙沙星、恩诺沙星、诺氟沙星、培氟沙星、沙拉沙星和双氟沙星6种氟喹诺酮类药物的检测限（LOD）均不超过0.5ng/mL,线性范围（IC20^IC80）在1.0¹2.1ng/mL之间;对虾、鳗鲡和鲫鱼三种水产样品中添加5.0、10.0和20μg/kg时,加标回收率为70.4%¹04.1%,相对标准偏差为5.0%¹4.7%;本方法可用于水产品中氟喹诺酮类（FQs）药物多残留的快速测定。      

测定食品中呋喃它酮代谢物3-氨基-5-吗啉甲基-2-恶唑烷酮的酶联免疫吸附法

目的建立以多克隆抗体为基础的测定食品中呋喃它酮代谢物3-氨基-5-吗啉甲基-2-恶唑烷酮(AMOZ)的间接竞争酶联免疫吸附分析法(ELISA)。方法以4-甲酰基苯氧基乙酸(4-FPA)为修饰剂,合成AMOZ半抗原衍生物,并使其分别与牛血清蛋白(BSA)和卵清蛋白(OVA)交联制备得到免疫原和包被抗原,经免疫动物(兔),获得抗NPAMOZ(AMOZ与衍生化试剂2-硝基苯甲醛所形成的结合物)的多克隆抗体。用酶联免疫分析法测定AMOZ(以NPAMOZ形式)。结果在包被抗原为2.5ng/mL,抗体为1:300000稀释,酶标二抗为1:10000稀释的优化条件下,ELISA测定AMOZ(以NPAMOZ形式)的IC50值(标准曲线中吸光度抑制至最大吸光度值50%时所对应的待测物浓度)为1.86ng/mL。抗体与呋喃它酮的交叉反应率较高(70.7%),与AMOZ的交叉反应率仅为0.32%,与其余三种硝基呋喃抗生素及它们的代谢物以及其它八种药物的交叉反应率很小(0.01%),表明抗体的特异性高。四种市售的肉样(鱼肉、虾肉、鸡肉、猪肝)用作加标实验,加标浓度分别为0.5、1.0和5.0ng/g,加标样品经衍生化处理后用ELISA测定,回收率为75.6%~112.2%,批间相对标准偏差(RSD)为8.3%~17.5%。结论本法具有高灵敏性和高特特异性,能用于动物产品中AMOZ的检测。     

北京市蔬菜中农药残留现状及慢性膳食暴露评估

目的了解北京市蔬菜中农药残留污染现状及对北京市居民的膳食风险水平。方法采集2013年北京市主要蔬菜生产基地和大型批发市场的蔬菜样品,采用NY/T 761-2008和GB/T 20769-2008等方法检测样品中的33种农药残留,分析北京市蔬菜中农药残留现状,并结合大城市居民的蔬菜消费情况和食品安全指数法对北京市居民从蔬菜中摄入农药风险进行慢性膳食暴露评估。结果 2013年北京市抽检的蔬菜中农药残留检测合格率达99.2%(502/506),农药超标的蔬菜为白菜类和绿叶菜类,拟除虫菊酯类农药检出频次较高,其构成比占40.8%,检出超标的农药为吡虫啉、毒死蜱、哒螨灵和百菌清;对所有检出农药进行慢性膳食暴露评估结果表明,百菌清的日摄入量相对较高,然而包括百菌清在内所有检出农药的食品安全指数值均小于1。结论北京市蔬菜中农药残留量的安全风险均在可接受范围内,百菌清是潜在的风险因子,应重点防患蔬菜中百菌清的残留风险。     

A sensitive and selective direct competitive enzyme-linked immunosorbent assay for fast detection of Sudan I in food samples

BACKGROUND: Sudan I, a synthetic azo dye, is considered to be a genotoxic carcinogen and is prohibited in foodstuffs for any purpose at any level worldwide. In this study, a sensitive and specific direct competitive enzyme‐linked immunosorbent assay (dc‐ELISA) for fast detection of Sudan I in food samples was developed for the first time. The monoclonal antibody against Sudan I was used as capture protein, while horseradish peroxidase labeled Sudan I conjugate prepared by the periodate method via ovalbumin (OVA) as a bridge was used as enzyme tracer. RESULTS: The standard curve of dc‐ELISA for Sudan I was constructed in the range 0.1–100 ng mL^?1 and the assay time was within 80 min. Sensitivity was 2.6 ng mL^?1 and the limit of detection was 0.08 ng mL^?1. Cross‐reactivity values of the assay with Sudan II, III and IV were 5.78%, 1.72% and 0.64%; no cross‐reactivity was found with six other edible colorants. The assay was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC₅₀. Recoveries of spiked Sudan I in five different samples including chilli powder, tomato sauce, hotpot seasoning and chilli sauce I and II were within 88.4–113.2% and the intra‐assay relative standard deviation was less than 14%. The dc‐ELISA was confirmed by conventional high‐performance liquid chromatography and the correlation coefficient of the two methods was 0.9902. CONCLUSION: The proposed dc‐ELISA method provides an alternative method for sensitive, specific and fast determination of Sudan I in food samples. Copyright © 2011 Society of Chemical Industry     

Development of a new monoclonal antibody based direct competitive enzyme-linked immunosorbent assay for detection of brevetoxins in food samples

Brevetoxin B (PbTx-2) was covalently linked to carrier protein bovine serum albumin and human gamma globulin. A monoclonal antibody against PbTx-2, which showed high cross-reactivity values with PbTx-1, PbTx-3 and PbTx-9 (more than 89%) was obtained from ascites and some characteristics of monoclonal antibody were studied. An direct competitive enzyme-linked immunosorbent assay (ELISA) for detection of PbTxs was developed, which showed an IC50 value of 5.3 ng mL⁻¹ with a detection limit of 0.6 ng well⁻¹. The recoveries of PbTxs from cockle (88.4%–102.3%) and oyster (89.4%–104.3%) demonstrated that the matrices of cockle and oyster where PbTxs are found do not interfere with the assay. The newly developed competitive ELISA appears to be a reliable and useful method for mass monitoring of PbTxs in mollusk.     

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

BACKGROUND: Imidacloprid is one of the main neonicotinoid insecticides widely used in agriculture owing its broad spectrum of activity and low bioaccumulation. However, imidacloprid is toxic to honey bees and other beneficial organisms, and its residues may occur in environmental and food samples, posing a potential hazard to consumers. In this study the imidacloprid derivative bearing a three‐atom length spacer was synthesized and coupled to carrier proteins. Highly sensitive and specific polyclonal antibodies against imidacloprid were successfully produced and the polyclonal antibody‐based enzyme‐linked immunosorbent assay (pAb‐ELISA) was developed. RESULTS: The ELISA standard curve was constructed within the concentration range 0.1–100 ng mL^?1. The IC₅₀ value for nine standard curves was in the range 1.2–3.0 ng mL^?1 and the limit of detection was 0.03–0.16 ng mL^?1. The sensitivity of the assay was one order of magnitude higher than that in most published papers. There was almost no cross‐reactivity of the antibody with four structurally related compounds (acetamiprid, nicotine, clothianidin and nitenpyram) and six other compounds, indicating that the assay displays not only high sensitivity but also high specificity. No detectable imidacloprid was found in 11 collected environmental and food samples by the assay. For imidacloprid‐spiked samples, acceptable recoveries of 73.4–94.4% and intra‐assay coefficients of variation of 2.2–12.8% were obtained. The assay was also validated with high‐performance liquid chromatography (HPLC) and a good correlation of ELISA with HPLC was achieved. CONCLUSION: The proposed ELISA provides a sensitive, specific, simple and cost‐effective quantitative/screening method for detecting imidacloprid in environmental and food samples. Copyright © 2011 Society of Chemical Industry     

Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle,chicken muscle,egg, fish,milk and kidney

A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1 μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5 μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10 μg/kg in kidney and 20 μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015 μg/kg, and the detection limits were 1.5 μg/kg in pig muscle and 6 μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r²) which was greater than 0.9.     

Validation of a multi-residue enzyme-linked immunosorbent assay for qualitative screening of corticosteroids in liver,urine and milk

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was applied for the qualitative screening analysis of dexamethasone, betamethasone, flumethasone, and prednisolone in milk and urine, and dexamethasone, flumethasone and prednisolone in liver samples at levels corresponding to the European Union maximum residue limit (MRL), or at required performance levels (RPLs) for substances for which there is no established MRL. Method validation was performed according to Commission Decision 2002/657/EC criteria established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CCβ), specificity, limit of detection (LOD), limit of quantitation (LOQ), recovery, within-laboratory reproducibility, linearity and ruggedness. LODs were 0.2, 1.2 and 0.6?µg?kg^?1 in milk, urine and liver samples, and LOQ values were 0.3, 1.2 and 1.4?µg?kg^?1 in milk, urine and liver, respectively. Recoveries from spiked samples ranged from 68% to 131% for dexamethasone, from 57% to 120% for flumethasone, from 60% to 155% for betamethasone, and from 23% to 32% for prednisolone, with a coefficient of variation (CV) between 1.6% and 21.2%. The CCβ value was below the MRL/RPL for all examined matrices. Moderate variations of some critical factors in the sample pre-treatment for liver and milk samples were deliberately introduced for ruggedness evaluation and did not result in any negative effects on corticosteroid detection. The proposed method is suitable for qualitative screening analysis of corticosteroids in the above-mentioned food in conformity with the current European Union performance requirements.     

Determination of atrazine and carbaryl pesticide residues in vegetable samples using a multianalyte dipstick immunoassay format

A dipstick assay for the simultaneous determination of atrazine and carbaryl in vegetable samples was developed. The analytical method involved a fast extraction procedure followed by a multi-strip membrane test. The assay took 10 min, with a detection limit (naked eye) of 10 and 200 µg l^-1 for atrazine and carbaryl, respectively. The cross-reactivities to related compounds tested were negligible except for propazine. Quantification of the pesticides was carried out by measuring the dot colour with a spectrophotometer. IC₅₀ values (inhibition constant at 50% of the maximum binding) of 2.04 μg l^-1 for atrazine and 92.8 μg l^-1 for carbaryl were achieved by this approach. Vegetable samples were extracted with MeOH. The proposed methodology was used to analyse atrazine and carbaryl and check for compliance with European Union maximum residue levels for vegetables. Recoveries (75-105%) were in agreement with those obtained by GC/MS or HPLC. Standard curves using 25% methanol/75% TBS were used for food sample assays using a multi-analyte dipstick. IC₅₀ values were 9.2 μg l^-1 for atrazine and 179.2 μg l^-1 for cabaryl.     

Development of a chemiluminescent enzyme‐linked immunosorbent assay for five sulfonamide residues in chicken muscle and pig muscle

BACKGROUND: An enzyme‐linked immunosorbent assay (ELISA) based on polyclonal antibodies with enhanced chemiluminescent (ECL) detection of sulfonamides in food samples has been optimised and characterised. The specificity of the assay was assessed by determining cross‐reactivities with a set of 16 sulfonamides. The aim of this study was to develop a method for determining sulfonamides with high sensitivity. RESULTS: The sensitivity of the developed ECL‐ELISA was higher than that of colorimetric ELISA. The sensitivities of five of the sulfonamides (sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine and sulfapyridine) ranged from 0.73 to 2.92 µg L^?1, with limits of detection of 0.10–0.43 µg L^?1. The coefficients of variation of intra‐assay and inter‐assay studies carried out over 5 days were mostly less than 10%. Recovery studies of chicken muscle and pig muscle were performed with simple and rapid extraction. Good recoveries (62.1–110.3%) were achieved and the results correlated well with those obtained using high‐performance liquid chromatography analysis. CONCLUSION: This study has provided an effective analytical technique for the rapid and reliable determination of residues of sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine and sulfapyridine in food samples with high sensitivity. To the authors' knowledge, this is the first report on chemiluminescent ELISA for sulfonamide analysis. Copyright © 2008 Society of Chemical Industry     

Application of enzyme-linked immunosorbent assay for quantification of the insecticides imidacloprid and thiamethoxam in honey samples

An enzyme-linked immunosorbent assay (ELISA) was used for the determination of residues of imidacloprid and thiamethoxam insecticides in honey after simple dilution of the samples without either extraction or cleanup. The ELISA enabled accurate determination of imidacloprid and thiamethoxam down to limits of 20 and 5 ng g^?1 in honey, respectively. Average recoveries of imidacloprid and thiamethoxam from the fortified honey samples were 90–120 and 96–122%, and coefficients of variation ranged 5–12 and 3–15%, respectively. The results from the ELISA agreed well with those by liquid chromatography–mass spectrometry (LC–MS) for the insecticide-spiked samples, with a correlation coefficient (r ²) of 0.96 and a regression coefficient (slope) of 1.03. The results indicate that ELISA is a suitable tool for the quantification of imidacloprid and thiamethoxam in honey.     

Reduction in residues of deltamethrin and fenitrothion on stored wheat grains by ozone gas

The effectiveness of ozone gas treatment in the removal of residues of organophosphate (fenitrothion) and pyrethroid (deltamethrin) pesticides in wheat grains by ozone gas was evaluated. The fenitrothion content was decreased following (60 μmol/mol) treatment after 180 min of exposure (a 66.7% reduction). At 20% mc (water activity a_w = 0.9). Similarly deltamethrin residues were reduced by 67.5, 88.1 and 89.8% after 60, 120 and 180 min of O₃ exposure, respectively. At the lowest mc and a_w (12% and 0.6) wheat grains had deltamethrin reductions of 80.6% and 85.7% after exposure for 120 and 180 min, respectively. The use of O₃ may be a potentially effective method of reducing pesticide residues in stored grains.     

酶联免疫吸附技术及其在食品安全检测中的应用研究进展

本文主要综述了酶联免疫吸附技术原理、分类及其在食品安全检测中的应用,并根据当前研究现状及存在问题进行了研究展望和提出几点发展建议.