兽药残留检测中酶联免疫试剂盒的选择原则与使用注意事项

畜产品中药物残留主要包括β-受体激动剂类、抗生素类、性激素类等药物。ELISA检测技术因其灵敏度高、特异性强、仪器设备简单、成本低、方法快速、简便等优点,成为畜产品中药物残留目前最理想的检测技术之一。为了考察试剂盒的各项指标与标示指标是否一致和能否正确使用试剂盒,本文就ELISA检测技术在畜产品中兽药残留检测试剂盒的选择原则和使用注意事项进行了归纳总结。     

王丽娜，郝利忠，韩春晓，苗翠

畜产品中药物残留主要包括β-受体激动剂类、抗生素类、性激素类等药物。ELISA检测技术因其灵敏度高、特异性强、仪器设备简单、成本低、方法快速、简便等优点,成为畜产品中药物残留目前最理想的检测技术之一。为了考察试剂盒的各项指标与标示指标是否一致和能否正确使用试剂盒,本文就ELISA检测技术在畜产品中兽药残留检测试剂盒的选择原则和使用注意事项进行了归纳总结。     

ELISA技术在畜产品安全检测中的应用

ELISA技术是针对畜产品进行全方位的安全检测,能够及时检测出畜产品自身的质量问题,从而针对其进行有效处理,这样能够有效保证投入到市场中的畜产品质量。ELISA技术的安全性较高,采用较少有机溶剂就能够针对畜产品进行合理检测,将其应用在当前的畜产品检测工作中,能够起到良好的效果。笔者主要是从ELISA技术的基本情况介绍入手,针对ELISA技术在畜产品安全检测过程中的有效应用进行全面细致的分析和说明。     

科技进展

饲料中喹乙醇ELISA检测试剂盒的研制及性能分析喹乙醇是一类广谱抗菌药,但由于能使动物发生中毒或死亡,还会在畜产品中残留,国家规定禁止用于家禽及水产养殖,只能限量用于体重低于35 kg猪的饲料。为了研制快速检测饲料中喹乙醇的ELISA试剂盒(OLA-Ki)t,该研究用喹乙醇单克隆抗体(OLA-mAb)建立间接竞争ELISA方法,     

对畜产品质量安全检测需求的思考

为贯彻落实《食品质量安全法》和《农产品质量安全法》,确保畜产品质量安全,新疆维吾尔自治区昌吉回族自治州畜产品质量检测中心开展了畜产品质量安全监督检测工作。检测样品包括肉、奶、蛋等食用畜产品及兽药、饲料、饲料添加剂等投入品,抽样对象主要是养殖场户、屠宰场、生鲜乳收购站、运输车、市场散奶、农贸批发市场等,检测指标主要是国家明令禁止的兽药残留、农药残留、污染物、违禁添加物及常规营养成分等。     

ELISA测定畜产品中克伦特罗残留量影响因子试验

采用ELISA(酶联免疫吸附法)测定畜产品中违禁药物克伦特罗残留量影响因子,试验结果表明,德国拜发与北京望尔生产的两种ELISA试剂盒具有操作简单、时间短、成本低、敏感度高等特点。在室温20～22℃条件下,试验制备过程中必须做到控温度、控转速、控流速及洗板过程中须注意一些事项,这样可大大提高在实际操作过程中检测的准确性。     

呕吐毒素检测测量系统分析

ELISA试剂盒法和上转发光法是检测呕吐毒素含量的两种常用方法，为确保检测数据的可靠性，本研究针对这两种检测方法进行测量系统分析。试验结果表明，两种测量系统分辨力均满足十分之一法则，但ELISA试剂盒法更为精确|稳定性、偏倚和线性均满足要求|重复性和再现性的实验中，ELISA试剂盒法R&R%为6.56%，P/T%为5.48%，均小于10%，表明该测量系统测量误差在可接受范围内|而上转发光法R&R%和P/T%均大于30%，不能满足要求。建议减少使用上转发光法检测样品中的呕吐毒素含量，试验结果以ELISA试剂盒法的检测结果更为准确、可靠。 [关键词]呕吐毒素|ELISA试剂盒法|上转发光法|测量系统分析     

动物性食品中β-受体激动剂类残留检测方法研究进展

食品安全是当今中国乃至世界普遍关注的问题,食品安全尤其以农产品中动物性食品最为引人注目。＂瘦肉精＂时间的影响,已引起足够的重视。畜产品质量安全检测,在畜产品安全中起无可替代的重要作用,是保障人们吃的放心的一项强有力手段。β-受体激动剂类残留检测,现已提到了相当高度,在畜产品残留检测占较大份额,检测方法发展也很快,相应快速检测卡、试剂盒产品开发也能基本满足检测需求。     

某品牌鸽新城疫抗体ELISA检测试剂盒性能评价

为检验市场上鸽新城疫抗体ELISA检测试剂盒的准确性及可靠性,从新疆喀什地区规模化鸽养殖场及鸽养殖合作社采集192份肉鸽血样,同时用血凝及血凝抑制试验以及某品牌鸽新城疫抗体ELISA检测试剂盒进行抗体检测,并以前者的检测结果作为"金标准",对ELISA检测试剂盒的性能进行评价。结果显示:ELISA试剂盒的敏感性为12.3%,特异性为97.4%,阳性似然比为4.73,阴性似然比为0.90,符合率为29.1%,约登指数为0.097,ROC曲线下面积为0.635。结果表明,该ELISA试剂盒检测鸽新城疫抗体的能力较差。这提示养殖企业及实验室选择ELISA方法检测鸽新城疫抗体前,应先对试剂有效性进行科学评估。     

浅析选择兽药残留ELISA试剂盒的几个注意事项

ELISA法(酶联免疫吸附法)是目前基层检测兽药残留应用最为广泛的筛选方法,具有分析效率高、实验室环境要求低、所需花费少等优点,但目前市场上试剂盒质量良莠不齐、参数繁多,购买人员对相关要求认识不足,直接影响了兽药残留监控效果。本文就试剂盒选择时的文件要求、试剂盒参数、操作简便性等方面进行了阐释,旨在使检测单位或人员能更好地选购兽药残留试剂盒,确保检测工作的顺利开展。     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。     

Research on the Production Process of Competitive ELISA Test Kit for Peste des Petits Ruminants Virus

In order to research on the production process of competitive ELISA test kit for peste des petits ruminants virus (PPRV), in this test, nucleoprotein (N) protein expressed in insect cell and monoclonal antibody of PPRV were used to establish a high specificity, sensitivity and strong competitive ELISA method for detection of PPRV, and the competitive ELISA procedures were optimized. 977 sheep and cattle serum samples collected from Xizang and Inner Mongolia were assayed with this established competitive ELISA and rC-ELISA antibody kit for PPRV of the OIE at the same time. The detecting results displayed that specificity, sensitivity and accuracy were 99.89%,93.82% and 99.38%. The competitive ELISA method of PPRV established in this assay would provide the basis for research on the production process of ELISA test kit as well as a reference for prevention scientifically.     

Study on Rapid Determination of Chloramphenicol Residue in Animal Products by High Sensitive ELISA

In this study, an ELISA method for determination of chloramphenicol (CAP) residue in animal products was used. According to the instruction of ELISA kit, CAP from pretreatment samples which was competed with CAP-HRP, and could be combined antibody coated. The content of CAP could be measured by color produced by substrate solution and HRP. The limit of the detection method in animal products was less than 0.1 μg/kg, and recoveries ranged from 74.4% to 105.4% for fortified samples at levels of 20 to 300 ng/kg with coefficient of variation values less than 15%.The ELISA kit could meet the requirement of detecting CAP residue in animal products.     

Utility of an in-office C6 ELISA test kit for determination of infection status of dogs naturally exposed to Borrelia burgdorferi

Serologic evaluation for the diagnosis of Lyme disease has been confounded by several factors, including a high prevalence of clinically normal dogs testing seropositive, persistence of antibodies, and the introduction of vaccines that will induce antibodies detectable by immunofluorescent antibody assay, whole-cell ELISA, and Western blot assay. The utility of a commercially available in-office test kit (SNAP 3Dx, IDEXX Laboratories) for the simultaneous detection of Borrelia burgdorferi and Ehrlichia canis antibodies and Dirofilaria immitis antigen was evaluated for its ability to detect exposure to B. burgdorferi in both vaccinated and unvaccinated dogs from a highly Lyme-endemic area of Connecticut. The test kit is an ELISA that uses a synthetic peptide (C6) that duplicates the sequence of the IR6 region. The in-office C6 ELISA kit was found to be particularly useful in Lyme-endemic areas because it can be used conveniently and reliably in the clinic to determine a dog's infection status regardless of the vaccination history of the animal.     

动物源性食品中莫能菌素残留ELISA试剂盒的研制

利用碳化二亚胺法合成抗原,进行动物免疫,制备特异性强的单克隆抗体。在筛选莫能菌素单克隆抗体的基础上,建立了莫能菌素ELISA检测方法,并研制出利用单克隆抗体对动物源性食品中残留的莫能菌素进行检测的试剂盒。本试剂盒的最低检测限为1μg/kg,试剂盒标准品变异系数为5.4%～13.1%,肌肉、肝脏样本的变异系数为5.1%～14.2%,肌肉和肝脏样本添加回收率分别为67.5%～87.4%和64.7%～86.7%。该方法的建立为莫能菌素的残留检测提供了可靠的分析检测手段。     

猪圆环病毒2型间接ELISA抗体检测试剂盒的研制及初步应用

以大肠杆菌原核表达系统表达的猪圆环病毒2型Cap蛋白为抗原,建立猪圆环病毒2型间接ELISA检测方法,优化ELISA反应条件,研制猪圆环病毒2型ELISA抗体检测试剂盒。与商品化试剂盒相比,该检测试剂盒敏感性、特异性和符合率分别为95.12%、92.86%和94.55%;同时与猪圆环病毒1型（PCV1）、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）等多种病毒阳性血清无交叉反应。试剂盒具有较好的重复性,在-20 ℃至少可保存1年以上,将其应用于临床血清样品的检测,结果安徽、广东、广西采样猪场的PCV2阳性率分别为100%、48.39%、100%。     

Comparison of commercial enzyme-linked immunosorbent assays and agar gel immunodiffusion tests for the serodiagnosis of equine infectious anemia

The purpose of this study was to estimate the performance characteristics (accuracy, detection limit, and precision) of commercially available enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) kits in comparison with a reference AGID kit for the detection of equine infectious anemia (EIA) antibodies in horses for regulatory use in Canada. A total of 285 positive and 315 negative samples by the reference AGID were tested blindly on 2 other AGID and 4 ELISA kits. Commercially available AGID kits for the serodiagnosis of EIA were found equivalent. The 3 ELISAs directed against antibodies to the p26 core protein also performed relatively well in comparison with the reference AGID, with excellent relative accuracy and acceptable precision. The single ELISA directed against antibodies to the gp45 trans-membrane viral protein yielded a lower relative sensitivity. The performance characteristics of the ELISAs directed against antibodies to p26 are, therefore, adequate to support the implementation of ELISA for regulatory purposes in Canada.     

几种检测猪弓形虫病ELISA诊断试剂盒的对比

以人工感染弓形虫的猪阳性血清及感染前的阴性血清为样品,比较3个国产(A、B、C)和2个国外(D、E)ELISA试剂盒检测猪弓形虫病的敏感性。用这5种ELISA试剂盒,检测人工感染弓形虫的猪阳性血清42份及感染前的阴性血清45份,并参考PCR扩增结果,比较这5种ELISA试剂盒检测猪弓形虫病的敏感性。5种试剂盒中,1种国产试剂盒的检测结果与PCR结果完全一致,其余4种试剂盒的检测结果都与PCR检测结果差别较大。结果表明,国产试剂盒A的检测效果最佳,国外试剂盒与部分国内试剂盒的检测结果相当,临床选用试剂盒时,可选择检测效果最佳的试剂盒A,不必盲目选择国外试剂盒。     

动物源性食品中泰乐菌素残留ELISA试剂盒的研制

采用杂交瘤技术,筛选并克隆出能够稳定分泌抗泰乐菌素（TYL）单抗的杂交瘤细胞株,免疫BALB／c小白鼠制备抗泰乐菌素单克隆抗体,建立泰乐菌素竞争ELISA检测试剂盒。结果表明,logit／log拟合标准曲线为y=-1．84x＋2．02,相关系数r=0．9944,线性检测范围为1．5～121．5ug／L,半数抑制浓度（IC50）为10.3ug／L,灵敏度为1．5ug／L,检测限为1．5～3．0ug／L;肌肉、肝脏、蜂蜜的添加回收率分别为64％～99％、61％～102％、60．5％～95％;平均批内和批间变异系数均小于10％;泰乐菌素与替米卡星（TIL）的交叉反应为40％,与其他类抗生素无交叉反应;此泰乐菌素试剂盒在4℃可保存6个月以上。本文建立的竞争ELISA检测方法可用于动物源性食品中泰乐菌素残留的定量检测。     

Reliability Evaluation of ELISA for Antibodies to ALV-A/B in Chicken Serum Samples

To evaluate the reliability of the positive rate of ALV-A/B specific antibodies detection in chicken serum and illustrate the stability and specificity problems existing in the commercialized Avian Leukosis Virus Subgroup A/B Antibody Test Kit, two batches of serum samples submitted by the company A which had no positive samples detected previously and the company B which had positive samples detected yet were tested using the same lot of ELISA kit by the professional operators. Then, among the positive samples detected for the first time, eight samples from company A and forty samples from company B whose S/P ratio all ranked higher were re-detected triply, and IFA were tested using the forty-eight samples above as first antibodies. The consistence of the results between ELISA and IFA assay was compared. The results demonstrated that, eight samples from company A turned into negative in the re-detection by ELISA kit, and IFA results were negative as well, which indicated stability problems existing in some batches of commercialized kit. Four samples (4/40) form company B turned into negative in the re-detection by ELISA kit, so as IFA results；thirty-six samples (36/40) form company B were positive in the re-detection by ELISA kit, however, twenty of them were IFA positive and sixteen of them were IFA negative, the complicated results indicated non-specificity detection were existed in some batches of commercialized kit except for the stability problem. Therefore, to achieve higher veracity, samples should be determined by both of ELISA and IFA in the massive detection.