Development of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Analysis of Diclazuril in Chicken Tissues

A highly sensitive and specific monoclonal antibody (Mab) against diclazuril was produced. The hapten with diclazuril coupled to diazotised 4-aminobenzoic acid was synthesized and conjugated to bovine serum albumin by the active ester method to form an immunogen for antibody generation. A novel diclazuril carboxymethyloxime derivative used in an ovalbumin conjugate was applied as a heterologous coating antigen and was expected to improve the immunoassay sensitivity. A sensitive and simple indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the Mab for the determination of diclazuril was developed. Under the optimized conditions, the icELISA for diclazuril showed a half maximum inhibition concentration (IC₅₀) value of 1.8 ng/mL, with limit of detection of 0.24 ng/mL and negligible cross-reactivities with other coccidiostat compounds including toltrazuril, robenidine, nicarbazin, halofuginone, amprolium, monensin, and maduramycin. The icELISA was successfully applied to diclazuril residue analysis in spiked chicken tissues. The average recoveries, intra-assay, and inter-assay coefficients of variation were in a range from 77.6 to 103.7 %, 3.7 to 13.0 %, and 5.6 to 18.3 %, respectively.     

酶联免疫检测技术在食品卫生理化检验中的应用

<正> 酶联免疫吸附法(ELISA)是日前分析化学领域中的前沿课题,特别是在食品和饲料中有毒有害物质的检测应用极为广泛。     

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.     

基于单克隆抗体的双酚A半抗原直接包被的酶联免疫吸附法的建立

传统的人工抗原包被酶联免疫吸附法(ELISA)依赖疏水相互作用将人工抗原与酶标板结合,会影响半抗原的呈现与识别,且多采用多克隆抗体为检测抗体,这些均会导致检测灵敏度下降和标准化难度增大。该研究选择双酚酸(BVA)作为双酚A(BPA)的半抗原与蛋白质偶联制备人工抗原免疫BALB/c小鼠,获得一株可分泌BPA单克隆抗体(MAb)的杂交瘤细胞株。利用3-氨丙基三乙氧基硅烷硅化处理酶标板,将BVA直接包被在酶标板上,建立了一种基于MAb的半抗原直接包被的BPA间接竞争ELISA法。该检测方法最低检测限IC10为0.29 ng/mL,半数抑制率IC50为5.4 ng/mL,水样中加标回收率为94.04%~102.31%。与人工抗原包被BPA ELISA检测方法(IC10:0.5 ng/mL,IC50:16.65 ng/mL,水样中加标回收率为89.72%~105.25%)相比,检测灵敏度显著性提高。     

酶联免疫法检测食品中乳酸链球菌素

建立了酶联免疫吸附法来检测乳制品、肉制品和含乳饮料中的乳酸链球菌素含量。在抗乳酸链球菌素多克隆抗体的基础上,建立间接竞争酶联免疫吸附法。所建立的间接竞争ELISA方法的灵敏度为0.1μg/mL,标准曲线范围为0.1～62.5μg/mL,乳制品的检测限为1.0μg/mL,肉制品的检测限为0.5μg/g,样本的添加回收率范围在80%～120%之间,变异系数<20%。所建立的间接竞争ELISA方法及制备的相关试剂盒具备准确、快速、简便的优点,能够满足乳制品、肉制品和含乳饮料中乳酸链球菌素含量的检测要求。     

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。     

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。     

Detection of Walnut Residues in Foods Using an Enzyme-Linked Immunosorbent Assay

ABSTRACT:  Tree nuts, including walnuts, can be responsible for allergic reactions. Food manufacturers have the responsibility to declare the presence of walnuts on packaged foods even when trace residues may be present from the use of shared equipment or the adventitious contamination of ingredients. The aim of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) method for the detection of walnut protein residues. Mixtures of raw and roasted English walnuts of several varieties were defatted, powdered, and used as separate antigens in sheep and New Zealand white rabbits. An ELISA was developed using the sheep antiroasted walnut serum as the capture reagent and rabbit antiroasted walnut serum as the detector reagent followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. The performance of the ELISA was validated by testing known amounts of walnut (0 to 100 ppm) either spiked into or manufactured into milk chocolate, cookies, muffins, or ice cream. Recoveries of 1 to 100 ppm walnut-in-chocolate ranged from 71.6% to 119%± 7% to 16.5%. The walnut ELISA has a detection limit of 1 ppm (1 μg/g) walnut in several food matrices. Substantial cross-reactivity was observed with pecan while minimal cross-reactivity was noted for hazelnut, mustard, mace, and poppy seed among almost 100 foods and food ingredients tested. This walnut ELISA can be used to detect undeclared walnut residues in foods and ingredients and as a tool to validate the effectiveness of allergen control programs for walnuts.     

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。     

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。     

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.     

Development of a Direct Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Fluoroquinolone Residues in Shrimp

Rabbit polyclonal antibodies were generated against norfloxacin, purified, and used as the basis of a direct competitive enzyme-linked immunosorbent assay for the screening of fluoroquinolones in shrimp. The developed method used a simple ethanol/acetic acid solvent extraction, which resulted in a 1.0-ng-norfloxacin/g limit of detection (based on the analysis of known negative and fortified shrimp samples). Norfloxacin extraction efficiencies were evaluated at two fortification concentrations and were greater than 70%, with an intra-assay variation less than 30%. The assay displayed greater than 10% cross reactivity against enro-, cipro-, sara-, and difloxacin. Incurred and known negative shrimp samples were analyzed and compared to the results obtained from an independent liquid chromatography tandem mass spectrometric method. All three instances in which fluoroquinolones were present at concentrations near or above the assay limit of detection (1.0 to 17 ng/g) were identified as positive by the newly developed assay, demonstrating the usefulness of this assay as a screening tool.     

Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Difenoconazole Residues in Fruits and Vegetables

An indirect, competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of difenoconazole was developed. Two haptens were designed and successfully synthesized. Hapten 1 had a particular moiety of difenoconazole, while hapten 2 had its full structure. The polyclonal antibodies against hapten-protein conjugates were prepared by immunizing rabbits. After optimization of the conditions, the detection limit (IC₁₅) and sensitivity (IC₅₀) were 4.58 and 29.10 μg L^?1, respectively. The cross-reactivities of the antibody with 11 triazole fungicides were all less than 0.1%, which showed that the antibody had excellent specificity. The recoveries of difenoconazole from the spiked samples ranged from 89.70 to 102.31% with good accuracy. The matrix effect was easily removed using a simple, rapid, and efficient extraction method on fruits and vegetables. The detection limit was all 229 μg kg^?1 in fruits and vegetables. To validate the ic-ELISA, samples were spiked with difenoconazole at three different concentrations and simultaneously analyzed using high-performance liquid chromatography (HPLC). The results showed a good correlation between the ic-ELISA and HPLC data (R ² = 0.9970). As a result, the developed immunosorbent assay is suitable for the quantitative determination of difenoconazole in fruits and vegetables.     

酶联免疫吸附(ELISA)法在食品微生物检测中的应用

介绍了酶联免疫吸附(ELISA)分析的原理及方法，并就其在食品微生物检测中的应用作了较详细的描述。     

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 of a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Buckwheat Residues in Food

Abstract: Buckwheat is a pseudocereal (an eudicot with seed qualities and uses similar to those of monocot cereals, family Poaceae) that is consumed in some Asian countries as a staple, and in some western countries as a health food. Allergic reactions to buckwheat are common in some countries. The objective was to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to detect traces of buckwheat that might inadvertently contaminate other foods in order to assure accurate labeling and consumer protection. Buckwheat-specific antibodies produced in 3 species of animals were tested for specificity and titer by direct ELISA and immunoblot. A sandwich ELISA was developed utilizing pooled rabbit antibuckwheat sera to capture buckwheat proteins and pooled goat antibuckwheat sera, followed by enzyme-labeled rabbit antigoat immunoglobulin G (IgG), to detect bound buckwheat proteins. The lower limit of quantification (LOQ) of the sandwich ELISA was 2 parts per million (ppm) of buckwheat in the presence of complex food matrices. The ELISA is highly specific with no cross-reactivity to any of 80 food ingredients and matrices tested. Validation studies conducted with buckwheat processed into noodles and muffins showed greater than 90% and 60% recovery, respectively. The percent recovery of buckwheat from noodles was similar to that achieved with a commercial buckwheat ELISA kit (ELISA Systems Pty. Ltd., Windsor, Queensland, Australia) at high buckwheat concentrations. However, the sensitivity of this ELISA was greater than the commercial ELISA. This newly developed ELISA is sufficiently specific and sensitive to detect buckwheat residues in processed foods to protect buckwheat-allergic subjects from potential harm. Practical Application: Buckwheat is becoming a common food ingredient in a number of processed foods due to potentially beneficial nutritional properties, without the celiac disease inducing glutenin proteins of wheat and related cereals. However, buckwheat causes allergy in some individuals and must be labeled and tested accurately to protect those with allergy to buckwheat. We describe the development of a new test assay to help food producers ensure that buckwheat is not present in foods that are not intended to contain buckwheat.     

酶联适体分析法检测葡萄酒中的赭曲霉素A

建立了竞争取代酶联适体分析方法,检测葡萄酒中的赭曲霉素A(OTA)。核酸适体和OTA特异性结合导致与核酸适体杂交的短链DNA解链,解离的DNA作为捕获元素,进一步特异性结合辣根过氧化物酶(HRP),HRP催化四甲基二苯胺(TMB)底物显色,测定A450nm与浓度的线性关系,确定OTA的检出限。考察了DNA包被浓度、杂交温度和封闭液等因素对检测的影响。结果表明,在优化的条件下,所建立的竞争取代酶联适体分析法对OTA检测有高灵敏度,检测限0.88μg/L,线性范围1~100μg/L在葡萄酒中添加时,加标回收率为92.03%~106.6%,相对标准偏差RSD(n=5)小于2.1%,此方法可用于葡萄酒中OTA的快速测定。     

玉米赤霉烯酮单克隆抗体制备及免疫分析

采用活泼酯法将玉米赤霉烯酮(Zaralenone,ZEN)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成免疫原ZEN-BSA和包被原ZEN-OVA,经紫外扫描和聚丙烯酰胺凝胶电泳鉴定后免疫BALB/c小鼠;挑选产生高效价和高敏感性抗体的小鼠进行抗原超强免疫,取脾细胞与SP2/0细胞融合获得1株稳定分泌抗玉米赤霉烯酮单克隆抗体的杂交瘤细胞(3D8),经鉴定,该抗体亚类为IgG1,轻链为k型。经体内诱生腹水并纯化获得效价为1∶2.048×106的单克隆抗体。建立了基于单克隆抗体的ZEN间接竞争ELISA法(ic-ELISA),该方法IC50和检出限分别为22.89pg/mL和10.07pg/mL。与α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、α-玉米赤霉醇和β-玉米赤霉醇的交叉反应率分别为95%、8%、12%、6%和5%,而与黄曲霉毒素B1、呕吐毒素、伏马毒素B1和赭曲霉毒素A未见有交叉反应。在玉米、大麦、小麦和燕麦中加标的回收率在86.4%~104.8%之间。该方法灵敏特异,可作为快速检测谷物中玉米赤霉烯酮的初筛方法。     

Rapid Detection of Four Organophosphorous and Neonicotinoid Toxicants Using Bi-enzyme Tracer Competitive Enzyme-Linked Immunosorbent Assay

A bi-enzyme tracer direct competitive enzyme-linked immunosorbent assay (dc-ELISA) based on two generic antibodies was developed. The effects of several physicochemical factors, such as heterologous antigen–horseradish peroxidase (HRP), methanol concentration, ionic strength, and pH value, were optimized to obtain satisfactory sensitivity of the assay. Under the optimized conditions, the 50 % inhibition concentration (IC₅₀) value for parathion, parathion-methyl, imidaclothiz, and imidacloprid was 57.28?±?2.73, 169.82?±?5.64, 52.48?±?3.46, and 53.08?±?2.05 μg L^?1, with a limit of detection (LOD, IC₁₀) of 0.56, 3.16, 0.62, and 0.51 μg L^?1, respectively. There was no obvious cross-reactivity (CR) with most of the neonicotinoids and organophosphorus pesticides. The recoveries of parathion, parathion-methyl, imidaclothiz, and imidacloprid in environmental and agricultural samples, including river water, soil, and cabbage, ranged from 82.54 to 116.29 %. The relative standard deviation (RSD) ranged from 1.59 to 8.09 %. The ELISA results were confirmed by gas chromatography (GC) with a high correlation coefficient of 0.9882. These results showed that the bi-enzyme tracer competitive enzyme-linked immunosorbent assay could be used as a sensitive tool for monitoring parathion, parathion-methyl, imidaclothiz, and imidacloprid in environmental samples and agricultural products.