Development of a Competitive Indirect Enzyme-Linked Immunosorbent Assay for Screening Phenylethanolamine A Residues in Pork Samples

Phenylethanolamine A (PEA), a new alternative β-agonist, has been illegally used in farming to promote the muscle growth in food-producing animals. In this study, a sensitive and convenient competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for determination of PEA residues in pork samples. The produced antibody was highly specific to PEA and exhibited a negligible cross-reactivity toward some other β-agonists. The developed technique was characterized by the limit of detection below 0.08 μg kg^?1 and the IC₅₀ value of 0.93 pmol mL^?1 (0.32 ng mL^?1). Validation of the technique was done using artificially spiked and naturally contaminated pork samples. The recoveries ranged from 79.6 to 112.6 % for the samples spiked at levels of 0.1–5 μg kg^?1 with the variation coefficients below 15 %. The analysis of naturally contaminated samples showed that the obtained data corresponded with the data obtained by the LC-MS/MS. The developed ciELISA was shown to be a feasible highly sensitive and specific screening tool for PEA residue analysis.     

Development of a Highly Sensitive Competitive Indirect Enzyme-Linked Immunosorbent Assay for Detection of Acrylamide in Foods and Water

Acrylamide (AA) has been classified as a probable human carcinogen and forms in certain foods, particularly plant-based foods that are rich in carbohydrates and low in proteins, during processing or cooking at high temperatures. In this study, polyclonal antibodies were raised against a hapten derived from acrylamide and 3-mercaptobenzoic acid (3-MBA). An indirect competitive enzyme-linked immunosorbent assay was developed to rapidly quantify AA in complex food matrices and water. The assay was very specific to the AA-3-MBA derivative and showed no cross-reactivity to asparagine, the main precursor of AA formation in foods, aspartic acid, AA, or 3-MBA. The assay was very sensitive with a limit of detection of 5.0 ng/g in model for food matrices to 0.1 μg/L in water. Good recoveries for AA were observed in all matrices tested, and the results using this method were comparable to those obtained from mass spectrometry methods including Food Analysis Performance Assessment Scheme control samples and results for different food products.     

A Highly Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) by Antigen Coating for Diethyl Phthalate Analysis in Foods

As we have known, with the plasticizer disturbance in 2011 in Taiwan, long-term exposure to diethyl phthalate (DEP), one of the widely used phthalate esters, can lead to serious health problems. Therefore, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) by antigen-coated plate format for DEP in foods was proposed in this paper. The polyclonal antibodies were raised against diethyl 4-aminophthalate (4-DEAP) conjugated to bovine serum albumin by the amino diazotization linkage method. Coating antigen was prepared with 4-DEAP conjugated to ovalbumin using the same procedure. Under the optimal experimental conditions, the ic-ELISA has a linear working range of 0.005–18.6 ng/mL (R ²?=?0.9921), with a limit of detection of 0.0049 ng/mL. Low cross-reactivity (<9 %) to structurally related phthalates was observed. The method was successfully applied to the determination of DEP in fruit juice, milky tea, pure milk, and sour milk, without purification or preconcentration. Satisfactory recoveries were obtained ranging from 91.1 to 109.3 %. The results suggested that the developed ic-ELISA is a simple, sensitive, and specific method for the rapid monitoring of DEP in food samples.     

A Highly Sensitive Aptasensor for Sulfamethazine Detection Using an Enzyme-Linked Aptamer Assay

To detect sulfamethazine (SMZ) residues in edible animal products, a colorimetric aptasensor that is based on indirect competitive enzyme-linked aptamer assay (ELAA) is developed in this study. This ELAA uses SMZ-specific aptamers as recognition elements and enzyme as the signal readout element. The detection sensitivity and the specificity of the ELAA were investigated. Our results showed that the indirect competitive ELAA had a ultrasensitivity with a low detection limit of 0.05 ng mL^?1. In addition, the proposed indirect competitive ELAA exhibited an 50% inhibition concentration value of 0.64 ng mL^?1 for SMZ, and recoveries up to 80.5–92.3% with coefficients of variation of less than 10%. Furthermore, when the ELAA was used for incurred samples by detecting SMZ in chicken muscles, the results obtained by the ELAA methods showed a good agreement with those obtained by established TRFIA and HPLC methods. These results demonstrate that the proposed indirect competitive ELAA can be efficiently used with good accuracy and precision, providing a simple method for the detection of SMZ residue in animal tissues.     

酶联免疫分析在调味料安全质量检测中的应用

酶联免疫分析是一种基于特异抗原抗体的反应、具有灵敏、简便而且成本低廉的免疫分析技术.本文对这种技术进行概述,详细评述该技术在调味料污染物检测中的应用,包括微生物污染以其生物毒素残留,违禁有机化合物污染和食品过敏原残留等;同时阐述了该技术在调味料质量安全监控的应用前景.     

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for the Determination of Thiacloprid in Agricultural Samples

An enzyme-linked immunosorbent assay (ELISA) was developed based on a polyclonal antibody for the analysis of thiacloprid in agricultural samples. Thiacloprid hapten was synthesized and conjugated to bovine serum albumin to produce an immunogen and ovalbumin to produce a coating antigen. Polyclonal antibodies were obtained from immunized New Zealand white rabbits. Under optimal conditions (5 % methanol, 0.1 mol/L Na⁺, pH 5.5), the ELISA showed a 50 % inhibitory concentration (IC₅₀) value of 0.01 mg/L and a limit of detection (IC₁₀) of 0.47 μg/L. No obvious cross-reaction with the other structural analogs of neonicotinoid insecticides showed that the polyclonal antibodies had a high specificity for thiacloprid. The average recoveries from spiked water, soil, pear, and tomato were in the range of 80 to 119 %. The results of the ELISA were confirmed by high-performance liquid chromatography, and the correlation of the results from the two methods had a high correlation coefficient of 0.99 (n?=?3) in spiked samples (soil, pear, and tomato). The proposed ELISA could successfully be applied to the determination of thiacloprid residues in agricultural samples.     

Development and Validation of a Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Screening of Florfenicol and Thiamphenicol in Edible Animal Tissue and Feed

To monitor the illegal use of florfenicol (FF) and thiamphenicol (TAP) in edible animal tissue and feed, a sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed with simple sample preparation and cleanup. The obtained monoclonal antibody (5F4) that has isotype IgG1 showed an IC₅₀ value of 0.21 μg L^?1 for FF and 0.35 μg L^?1 for TAP, respectively. It did not exhibit measurable cross-reactivity with other antibiotics. The limits of detection (LODs) for FF and TAP in a muscle matrix ranged from 0.07 to 0.14 μg kg^?1 and in a feed matrix ranged from 2.9 to 5.2 μg kg^?1. The recoveries were 72.8 to 113.4 % with a coefficient of variation of less than 15 %. Good correlation between the ELISA and HPLC-MS/MS results in the tissues tested demonstrated the reliability of our ic-ELISA. This ELISA is a useful tool for screening FF and TAP in edible animal tissue and feed.     

Enzyme-Linked Immunosorbent Assay for Detection of Mold in Tomato Puree

A double-sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of molds (Ahernaria alternata, Geotrichum candidum and Rhizopus stolonifer) in tomato puree by the use of antisera raised in rabbits injected with homogenates of the lyophilized boiled molds. Cross reactivity among the three species was less than 10%. Detection limits were approximately 1μg dried mold/g of sample, a sensitivity greater than that of most chemical methods. Positive relationships between ELISA readings and the amounts of mold added to puree were observed, while background ELISA values for puree controls were negligible.     

绿色无毒黄曲霉毒素B_1免疫检测方法

建立了一种基于抗独特型抗体的绿色无毒的ELISA方法,并用于检测面粉中黄曲霉毒素B1。通过酶解2种抗黄曲霉毒素小鼠单克隆抗体(11A9和1G3)制备F(ab’)2片段,并将其免疫新西兰大白兔制备抗独特型抗体。最终得到两种抗独特型抗体,并对抗独特型抗体和AFB1进行相关性分析。通过实际样品添加回收,其批内回收率为115.60%~121.88%,变异系数在5%以内;而批间回收率为111.89%~126.98%,变异系数低于8%。分析结果准确可靠,表明此无毒绿色ELISA是一种安全可靠的AFB1分析方法。     

沙丁胺醇直接酶联免疫快速检测的方法

沙丁胺醇与瘦肉精同属于禁用的饲料添加剂,可以在动物体内大量残留,人体过量摄入会引起中毒反应。为了建立沙丁胺醇的快速检测方法,从硫酸沙丁胺醇出发制备了半抗原沙丁胺醇丁二酸衍生物,用混合酸酐法将半抗原与载体蛋白-钥孔嘁血蓝蛋白(KLH)偶联作为免疫抗原制备了沙丁胺醇的多克隆抗体,建立了沙丁胺醇直接竞争酶联免疫检测方法,该方法抗体最佳包被抗体量为1μg/孔,酶标抗原稀释比例为1∶16000,掩蔽剂采用脱脂奶粉。所建立的方法具有很高的灵敏度和特异性I,C50为0.90ng/mLI,C15达到了0.05ng/mL,远低于国家残留限量标准,与沙丁胺醇的结构类似物基本没有交叉反应。     

雷洛昔芬抗体的制备及ELISA方法的初步建立

本文主要研究了雷洛昔芬抗体的制备和酶联免疫吸附测定(enzyme-linked immuno sorbent assay,ELISA)方法的初步建立。实验中采用丁二酸酐对雷洛昔芬进行衍生,合成了雷洛昔芬半抗原。采用碳二亚胺法,将雷洛昔芬半抗原与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成免疫原和包被抗原,并通过紫外光谱进行鉴定,结果显示雷洛昔芬与载体蛋白偶联成功。免疫大白兔制备了雷洛昔芬抗体,抗体效价达1.28×10~5,半数抑制浓度(half inhibit concentration,IC50)15.4μg/L,与其他类似抗雌激素药物没有交叉反应,说明所制备的抗体特异性好。通过优化抗原抗体反应浓度初步建立了雷洛昔芬ELISA方法,结果显示最佳抗原浓度为300μg/L,最佳抗体工作浓度为1:1.0×10~5,标准曲线在0.4~102.4μg/L范围内线性关系好,R~2=0.9853,最低检测能力0.4μg/L。本研究为进一步开发雷洛昔芬快速检测试剂盒提供了技术参考。     

雌二醇间接竞争酶联免疫吸附方法的建立

建立食品中雌二醇免疫分析方法。将雌二醇进行结构改造合成半抗原并经过核磁氢谱和红外光谱验证,再采用活泼酯法与载体BSA和OVA偶联制备人工抗原,免疫兔子制备多克隆抗体,经过反应条件优化建立间接竞争酶联免疫吸附检测方法。结果表明：IC50为3.9ng/mL,检测限为0.3ng/mL,交叉反应率均小于1.21%。检测方法具有高灵敏度和特异性,可应用于食品中雌二醇检测试剂盒的研制。     

一种检测杂环胺类化合物的酶联免疫检测方法的建立

为了制备一种广谱性杂环胺抗体,并建立一种可以实现多种杂环胺同时检测的快速分析方法。以杂环胺2-氨基-3,4-二甲基咪唑[4,5-f]喹喔啉(MeIQx)为原料,将其与丁二酸单甲酯酰氯(MCO)反应合成杂环胺半抗原,通过活化酯法将半抗原与蛋白偶联制备免疫原进一步制备多克隆抗体,最终建立间接竞争酶联免疫分析方法(ic-ELISA)。该方法灵敏度(IC₅₀,以MeIQx计)为81.16 μg/L,检测限(IC₁₅)为12.07 μg/L。方法对喹啉类杂环胺(IQ、MeIQ)、喹喔啉类杂环胺(IQx、MeIQx、4,8-DiMeIQx、7,8-DiMeIQx、4,7,8-TriMeIQx)以及吡啶类杂环胺(PhIP)具有相同的识别能力,交叉反应率均达到93%以上。油炸牛肉和肉松样品中杂环胺(MeIQx)添加回收率在91.18%~98.64%之间,检测结果与液相色谱串联质谱法(LC-MS/MS)有很好的一致性(R²=0.9927)。本文建立的酶联免疫分析方法可以实现喹啉类、喹喔啉类以及吡啶类杂环胺总量的检测,为热加工肉制品中杂环胺的检测提供了一种简单、准确的快速检测方法。     

呋喃西林代谢物酶联免疫吸附测定方法的建立及性能测定

建立动物源性食品中呋喃西林代谢物酶联免疫吸附测定方法。采用自制的呋喃西林代谢物抗原、抗体为基础原料,通过优化反应条件,建立间接竞争酶联免疫吸附试验(enzyme-linked immunosorbent assays,ELISA)检测方法,并对方法的性能进行测定。结果表明:建立的检测方法在0.03μg/L^2.43μg/L范围内呈线性,检测灵敏度为0.03μg/kg,鱼肉组织样本、虾肉组织样本的检测限分别为0.236、0.229μg/kg,回收率均在75%~120%之间,批内、批间变异系数均在15%以下,通过与标准方法液相色谱-串联质谱(high performance liquid chromatography-mass spectrum/mass spectrum,LC-MS/MS)对比验证,两种方法用于鱼肉样本的检测呈现较好的相关性,相关系数R2为0.9944。建立的呋喃西林代谢物酶联免疫吸附测定方法的灵敏度、准确度和精密度等参数均符合我国动物源性食品中兽药残留检测方法的规定。     

吡虫啉残留酶联免疫吸附检测方法的建立

通过化学修饰合成吡虫啉(imidacloprid,IMI)人工半抗原,采用碳二亚胺法(EDC)将该抗原与牛血清蛋白和卵清蛋白偶联成功制备分子结合比合理的免疫抗原(IMI-BSA)和包被抗原(IMI-OVA)。对经IMI-BSA免疫的6周龄Balb/c实验鼠的免疫抗血清进行间接酶联免疫吸附和阻断酶联免疫吸附,初步探明抗吡虫啉多克隆抗体(IMI-pAb)的免疫学特性。在此基础上,采用聚乙二醇(PEG)介导细胞融合技术进行了免疫鼠脾细胞和骨髓瘤细胞融合,通过阳性杂交瘤细胞筛选和克隆化培养,获得3C8杂交瘤细胞株。结果显示:该3C8杂交瘤细胞株具有高效价、高亲和力和强特异性的特点;通过方阵滴定法确定最佳抗原包被浓度和最佳抗体稀释倍数,建立基于吡虫啉单克隆抗体(IMI-mAb)的IMI残留阻断酶联免疫吸附,其线性范围为7.48×10-6～3.24×10-4mg/mL(R2=0.9928),最低检测限为8.00×10-6mg/mL。     

An Enzyme-Linked Immunosorbent Assay to Detect Salinomycin Residues Based on Immunomagnetic Bead Clean-up

Polyether antibiotics have been widely used for the treatment and prevention of coccidiosis in chicken farming. In the present study, an efficient, simple and inexpensive competitive indirect enzyme-linked immunosorbent assay (ciELISA) method based on immunomagnetic sample clean up was developed for salinomycin. Monoclonal antibodies were immobilized on the surface of carboxylic acid magnetic beads (2.8 μm in diameter). After a simple extraction, residues in sample extracts were specifically adsorbed and the supernatant removed by magnetic separation. Analytes retained on the beads were then released by elution prior to ciELISA. The limit of detection for salinomycin in chicken muscle and liver was 22 and 18 ng mL^?1, respectively, and the linear quantitative range was 47–653 ng mL^?1. Intra-assay recoveries ranged from 86.00 to 99.32%, and inter-assay recoveries were between 85.68 and 96.34%. The inhibition efficiency and sensitivity of this method was improved compared with traditional hydrophilic-lipophilic balance column clean up ciELISA. Furthermore, the convenience and repeatability of the immunomagnetic cleanup renders the new method of high value for the analysis of drug residues in complex matrices.

Open image in new window

Graphical Abstract The test method of schematic diagram

     

Substitution of Antibody with Molecularly Imprinted Film in Enzyme-Linked Immunosorbent Assay for Determination of Trace Ractopamine in Urine and Pork Samples

A new direct competitive enzyme-linked immunosorbent assay (ELISA) with molecularly imprinted film as artificial antibody was developed to detect ractopamine (RAC) in urine and pork samples. The imprinted film was directly synthesized on the well surface of 96-well plate by an organic–inorganic hybrid technology and evaluated by fourier transform infrared (FT-IR), static, and kinetic adsorption experiments. The imprinted film exhibited an antibody-like binding ability and rapid adsorption speed. The established ELISA method gave a good sensitivity (IC₅₀, 15.77 μg L⁻¹) and a low detection limit (IC₁₅, 0.01 μg L⁻¹) for RAC under the optimum conditions, and it was applied to the determination of RAC in urine and pork samples spiked at three levels with recoveries ranging from 77.7% to 108.9% (urine) and from 93.5% to 101.1% (pork). The obtained results, which correlated well with those gained from the traditional high performance liquid chromatography (HPLC) method, demonstrated that the developed ELISA method was reliable for RAC determination in real samples.     

Linking Solid Phase Extraction and Enzyme-Linked Immunosorbent Assay to Measure Chlorpyrifos in Fish Tissue

ABSTRACT: A method for measuring chlorpyrifos in fish tissue coupling solid phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) is described. Fish tissue was added to a silica gel column and extracted with hexane. The eluant was passed through an SPE column (C-18) and analyzed using ELISA. The method, which was comparable to an FDA GC/ECD method, gave recoveries from 83.4% to 87.5% for fortified fish samples, a lower limit of detection of 0.01 ppm, a linear range from 0.01 to 0.5 ppm, and correlation coefficients from 0.86 to 0.96. Nineteen contaminated fish samples analyzed using both ELISA and GC/ECD methods gave comparable chlorpyrifos concentrations with a correlation coefficient of 0.78.     

A Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for 5-Formyltetrahydrofolate Detection in Maize Kernels

Maize is one of the most important staple food crops worldwide. 5-Formyltetrahydrofolate (5-F-THF), the most dominant and stable folate derivative in maize kernels, is important to human health and acts key roles in several metabolic pathways and biological processes. To assist investigating maize germplasm and subsequent breeding of maize varieties containing high levels of 5-F-THF, a cost-effective and high-throughput methodology for 5-F-THF detection is required. In the present study, monoclonal antibodies (mAbs) against 5-F-THF were raised in mice, followed by establishment of an indirect competitive enzyme-linked immunosorbent assay (icELISA). The icELISA showed an IC₅₀ of 35.10 ng/mL and a working range of 7.90–263.89 ng/mL for 5-F-THF. The analytical recovery for 5-F-THF in the maize kernels with icELISA was 66–111 %, and that with liquid chromatography-mass spectrometry (LC-MS) was 57–114 %, indicating a good consistency between these two methods (Slope, 0.92; R ², 0.98). We conclude that the sensitive and versatile icELISA can be used for high-throughput detection of 5-F-THF in maize kernels.     

虾中硫丹残留的酶联免疫吸附检测方法的研究

目的:建立一种虾中农药硫丹残留的快速检测方法.方法:采用酶联免疫吸附试验(ELISA)测定农药硫丹残留.提取溶剂为丙酮.该检测方法中用阴性样品作标准曲线.结果:该方法的检出限(IC15)约为1.6 μg/L,灵敏度(IC50)约为17μg/L.线性范围1.8～60μg/L.平均回收率67.56%～99.81%.结论:该检测方法符合<日本肯定列表>中规定的水产品中硫丹的检测要求,且样品的处理过程较为简单,适用于大量样品的快速检测.