【作者】 蒋文晓；

【导师】 吴永宁； 沈建忠；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 近年来,动物性食品中的兽药残留问题引起了国内外的普遍关注。目前的免疫分析方法多数只能检测一种或一类具有相似结构的药物。为了提高检测效率、节省检测时间、降低检测成本,发展能够同时检测两种或两类兽药残留的免疫分析方法对于保障动物性食品安全具有较为重要意义。本文开展了同时检测两种喹噁啉类药物残留标示物以及同时检测磺胺类、喹诺酮类两类药物残留的免疫分析方法研究。以4-(3-甲基-喹嗯啉-2-甲酰胺基)丁酸为半抗原制备了单克隆抗体,该单抗对3-甲基-喹噁啉-2-酸(MQCA)和喹噁啉-2-羧酸(QCA)等5种喹嗯啉类药物双脱氧代谢物的交叉反应率在50%-2400%之间,其中MQCA和QCA的IC50值分别为4.8和9.6ng/mL。本文利用该抗体建立了检测动物组织(鱼肉、虾肉、猪肉和鸡肉)中MQCA和QCA残留的ELISA方法。该方法检测动物组织中MQCA和QCA残留的检测限(LOD)为1.54μg/kg,当MQCA和QCA在动物组织中添加浓度分别为2-20μg/kg时,该方法的添加回收率在76%-108%之间,变异系数在4.2%-13.3%之间,符合兽药残留检测的相关要求。为了提高免疫分析方法的灵敏度,本文建立了检测MQCA和QCA残留生物素亲和素信号放大酶联免疫分析(BA-ELISA)和化学发光免疫分析(CLIA)。BA-ELISA方法对MQCA和QCA的IC50值为1.6和3.6ng/ml;检测动物组织的LOD值为1.04μg/kg;MQCA在动物组织中的添加浓度在2-8μg/kg时,添加回收率在72%-106%,变异系数在6.7%-12.6%之间。CLIA方法对MQCA和QCA的IC50值为0.42和1.05ng,mL;检测动物组织的LOD值为0.76μg/kg;MQCA在动物组织中的添加浓度为1-4μg/kg时,添加回收率在71%-108%之间；变异系数在7.0%-14.4%之间。上述两种方法的灵敏度和检测限等符合兽药残留检测的相关要求。以辣根过氧化物酶HRP标记羊抗兔二抗来识别磺胺类药物(SAs)的多克隆抗体,以碱性磷酸酶ALP标记羊抗鼠二抗来识别喹诺酮药物(QNs)单克隆抗体,从而实现在同一个反应体系中同时检测牛奶中22种SAs和13种QNs残留的双显色酶联免疫吸附试验(DC-ELISA)。该DC-ELISA方法对SAs和QNs的检测限分别为5.8和2.4μg/L。当SAs和QNs在牛奶中的添加浓度分别为10-100μg/L时,SAs的添加回收率均在67%-101%之间,变异系数在8.1%-16.4%之间；QNs的添加回收率在72%-105%之间,变异系数在4.8%-9.3%之间,该方法满足SAs和QNs两类兽药残留检测的相关要求。以琼脂糖凝胶作为载体,建立了同时检测牛奶中14种SAs和13种QNs的凝胶穿流免疫亲和层析试验(FTIACT)方法,并评价了HRP、荧光微球、量子点、脂质体包裹量子点(LQDs)四种标记物的试验效果。以LQDs做标记物的FTIACT肉眼判断检测牛奶中SAs和QNs残留的Cut off值分别为1和0.5μg/L。通过光密度值分析,LQDs-FTIACT定量分析方法的LODs为0.27μg/L(SAs)和0.12μg/L(QNs),在空白牛奶样品中SM2添加浓度为0.25、0.15μg/L和CIP的添加浓度为0.1、0.25μg/L时,平均添加回收率在89%-114%之间,变异系数在9.2%-12.3%之间,满足兽药残留检测的相关要求。更多还原

【Abstract】 The veterinary drug residues in animal-origin foods caused widespread concern at home and abroad in recent years. Nowadays, most of the immunoassays focus on detecting one analyte or one class of structural-related analytes. In order to improve dtermination efficiency, decrease detecting time and decrease expenses, it is urgent to develop multi-analyte immunoassays. In this paper, we developed immunoassays for rapid detecting two marker residues of quinoxaline and immunoassays for detecting two class of veterinary drugs (SAs and QNs)In this paper,4-(3-methylquinoxaline-2-carboxamido)butanoic acid was used as hapten for monoclonal antibody production. The produced MAb showed good cross reactivities (50%-2400%) with the metabolites of quinoxaline, and the IC50values of QCA and MQCA were4.8and9.6ng/mL. Based on this MAb, a broad specificity ELISA was developed for simultaneously detecting MQCA and QCA in animal edible tissues (fish, shrimp, pork and chicken). The limit of detection was1.54μg/kg for MQCA and QCA in animal edible tissues. Satisfying recovery ranged from76%-108%was obtainded with the coefficients of variance ranged from4.2%to13.3%, when the samples were spikded with MQCA (2,4,8μg/kg) and QCA (4,10,20μg/kg). All these results meet the requirement of residue analysis.To improve the sensitivity of immunoassay, BA-ELISA and CLIA were debeloped for MQCA and QCA residues analysis. The IC50of BA-ELISA was1.6and3.6ng/mL for MQCA and QCA in buffer, and the LOD was1.04μg/kg in animal edible tissues (fish, shrimp, pork and chicken).When the blank samples were spiked with MQCA at2,4,8μg/kg, the intra-assay and inter-assay recoveries were in the range of72%-106%, and the corresponding CV values ranged from6.7%to12.6%. The IC50of CLIA was0.42and1.05ng/mL for MQCA and QCA in buffer, and the LOD was0.76μg/kg. When the fish sample were spiked at1,2,4μg/kg, the intra-assay and inter-assay recoveries were in the range of71%-108%, with the CV values ranged from7.0%to14.4%. The detection limit and sensitivity of the immunoassays meet the requirements of the legislation of EU, US and China.In this study, a novel DC-ELISA was developed for simultaneously detecting22SAs and13QNs in milk. The DC-ELISA using ALP labeled goat anti-mouse IgG to recognize anti-QN MAb, and using HRP labeled goat anti-rabbit IgG to recognize anti-SA PAb. In this way, the DC-ELISA could be used for simultaneous detecting SAs and QNs in one well. The method for SAs and QNs detection limits were5.8and2.4ng/mL. When the milk samples were sipked with SAs and QNs (10,50,100ng/mL), the mean recoveries were in the range of67%-101%for detecting SAs residues in milk, and the corresponding CV values ragned form8.1%to16.4%. The mean recoveries were in the range of72%-105%for detecting QNs residues in milk, and the corresponding CV values ranged from4.8%to9.3%. All these results meet the requirement of residue analysis.In this paper, we used agrose as the carrier of the immune-reaction and developed a novel FTIACT method for simultaneously detecting14SAs and13QNs. In this paper, we tested four different labels (horseradish peroxidase, fluorescent microspheres, quantum dots, lipsome encapsulated quantum dots) and their application in FTIACT. The visual detection limits of the LQDs-FTIACT method were1and0.5ng/mL. The OD values were processed and analyzed from the image of the FTIACT results using Image Pro Plus6.0software, and the OD values were used to develop a standard curve for FTIACT quantitative analysis. When using LQDs as labels, the detection limit of the FTIACT method were0.27ng/mL (SAs) and0.12ng/mL (QNs). The mean recovery ranged from89%to114%, and the CV values ranged from9.2-12.3%when the blank milk samples were spikded at0.25,0.15μg/kg for SM2and0.1,0.25μg/kg for CIP. All these results meet the requirement of the legislation and indicated that the FTIACT method could be used for simultaneously screening SAs and QNs in milk.更多还原