基于单链抗体的牛奶中21磺胺和20氟喹诺酮类药物残留生物发光免疫分析方法研究

发布时间：2018-03-26 10:40

  本文选题：磺胺类药物　切入点：氟喹诺酮类药物　出处：《中国农业大学》2016年博士论文

【摘要】：动物性食品中化学性危害物的残留问题是食品安全重要的组成部分,发展高效、灵敏、快速、多残留的免疫分析方法是保障食品安全的有效途径。免疫分析中传统的多克隆抗体和单克隆抗体,一经制备,后期改造的可能性很小。而重组抗体可从基因水平上对抗体进行修饰、改造、定向标记、体外表达,而且其单价结合特性,灵敏度更高。荧光素酶由于其发光强度高,发光迅速,适合于原核表达,以其为基础的生物发光技术由于灵敏度高,光谱范围广等优点在兽药残留检测领域已经引起关注。本论文开展了动物性食品中磺胺类和氟喹诺酮类药物残留的生物发光免疫分析方法研究,旨在为兽药残留监控提供快速、灵敏、高通量的检测手段。以1：1融合表达的方式分别将碱性磷酸酶(Alkaline phosphatase, ALP)和海肾荧光素酶(Renilla luciferase, Rluc)与氟喹诺酮类药物(Fluoroquinolones, FQs)的单链抗体(single chain variable fragment, scFv)进行定向标记,构建了FQs-scFv-ALP和FQs-scFv-Rluc融合蛋白。分别建立了可检测20种FQs的化学发光直接竞争免疫分析方法(CL-cdELISAFQs-scFv-ALP)和可检测20种FQs的生物发光直接竞争免疫分析方法(BL-cdELIS AFQs-scFv-Rluc)。以诺氟沙星(Norfloxacin,NOR)分别建立标准曲线,CL-cdELISAFQs-scFv-ALP中NOR的IC50值为0.15μg/L,线性范围为0.04-1.08μg/L,牛奶样品采用乙酸乙酯提取,氮吹,PBS复溶的方法处理后可消除基质效应,检测限(Limit of detection, LOD)为 0.021μg/L,与其他19种氟喹诺酮类药物的交叉反应率介于0.43-133.89%; BL-cdELISAFQs-scFv-Rluc中NOR的IC50值为0.031 μg/L,线性范围为0.006-0.16μg/L,牛奶样品采用直接稀释40倍的方法可消除基质效应,LOD为0.124μg/L,与其他19种氟喹诺酮类药物的交叉反应率介于0.45-129.7%。以欧盟规定牛奶中必检的五种氟喹诺酮类药物,环丙沙星(Ciprofloxacin, CIP)、恩诺沙星(Enrofloxacin, ENR)、马波沙星(Marbofloxacin, MAR)达氟沙星(Danofloxacin, DAN)以及氟甲喹(Flumequine, FLU)进行添加回收试验,其中CL-cdELISAFQs-scFv-ALP的批内和批间添加回收率分别为78.2-119.4%和79.6-117.4%,变异系数分别为3.1-11.4和4.4-10.3%。BL-cdELISAFQs-scFv-Rluc的批内和批间添加回收率分别为78.4-117.2%和77.8-117.9%,变异系数分别为6.9-13.4%和7.4-12.9%。对比结果显示,本研究建立的两种方法的IC50值比传统ELISA方法分别降低了约2倍和10倍,生物发光的IC50值比化学发光的IC50值低大约5倍,而且耗时短,牛奶样品处理简单,虽然生物发光的变异系数大于化学发光,但是回收率均介于75-120%,变异系数小于15%,均达到了兽药残留分析的要求。构建了磺胺类药物(S ulfonamides, SAs)单链抗体与萤火虫荧光素酶(Firefly luciferase, Fluc)的融合蛋白(SAs-scFv-Fluc)。基于FQs-scFv-Rluc 和 SAs-scFv-Fluc,建立了同时检测牛奶中21种磺胺类和20种氟喹诺酮类药物的多残留直接竞争生物发光免疫分析方法(DBL-cdELISA)。以NOR和磺胺二甲基嘧啶(Sulfamethazine, SMZ)分别建立标准曲线,NOR和SMZ的IC50值分别为0.051μg/L和0.211μg/L。与其他19种氟喹诺酮类药物的交叉反应率介于0.56-130.46%,与其他20种磺胺类药物的交叉反应率介于2.25-1406.67%。牛奶稀释80倍可消除基质效应,NOR和SMZ的LOD分别为0.232μg/L和1.894μg/L。分别选取五种氟喹诺酮类药物(CIP, ENR, MAR, DAN以及FLU)和五种磺胺类药物(SMZ,磺胺二甲氧嘧啶(Sulfadimethoxine, SDM),磺胺喹VA林(Sulfaquinoxaline, SQX),磺胺间甲氧嘧啶(Sulfamonomethoxine, SMM)以及磺胺甲基异VA唑(Sulfamethoxazole, SMX))进行添加回收试验。五种FQs的添加回收率为76.6-118.4%,变异系数为5.7-9.7%五种SAs的添加回收率为79.2-118.6%,变异系数为5.4-9.6%,满足兽药残留检测的相关要求。双荧光素酶的使用可使底物同时加入,信号同时采集,实现了基于双酶标记的固相免疫分析方法中两类物质的同时检测。以海肾荧光素酶和腔肠素为能量供体,量子点为能量受体,氟喹诺酮类药物为模式药物,建立了基于单链抗体的生物发光能量共振转移(QD-BRET)均相免疫分析体系。海肾荧光素酶的最适底物为腔肠素h, QD-BRET的福斯特距离为7.86nm,供体与受体之间的距离为8.9nm。以ENR建立标准曲线,其IC50值为0.782μg/L,线性范围为0.023-25.60μg/L, LOD为2.54 ng/L。与7种代表性的氟喹诺酮类药物的交叉反应率介于2.55-111.6%,与第二章第三章的交叉反应率一致。五种氟喹诺酮类药物(CIP、ENR、MAR、DAN以及FLU)批内和批间的添加回收率分别为78.5-118.2%和79.3-116.6%,变异系数均小于10%。虽然本方法灵敏度不及DBL-cdELISA,但本方法是一个均相的分析方法,省去了包被、洗涤等步骤,耗时短,操作简单,并且可以满足氟喹诺酮类药物的残留检测要求。综上所述,本研究建立的基于单链抗体和荧光素酶融合蛋白的均相和非均相生物发光免疫分析方法均可用于牛奶中磺胺类和氟喹诺酮类药物的残留检测,在最大残留限量之下可检测21种磺胺类药物和20种氟喹诺酮类药物。与化学发光相比,生物发光灵敏度高,耗时短,且牛奶样品处理简单,不同荧光素酶的联合应用,可实现多种物质的同时检测。此外本研究初步证明,基于量子点的QD-BRET体系可用于小分子物质的检测,可作为兽药残留监控中快速、灵敏、高通量的检测手段。
[Abstract]:The residual chemical hazards in animal food is an important part of food safety, the development of efficient, sensitive, rapid, multi residue immunoassay method is an effective way to ensure food safety. The traditional immunoassay of polyclonal antibodies and monoclonal antibodies, the preparation, the possibility of reform is very small. The recombinant antibody can be modified, the antibody from gene level transformation, directional marker, and its expression in vitro, monovalent binding properties, higher sensitivity. Luciferase because of its high luminous intensity, luminous rapidly, suitable for prokaryotic expression, based on the bioluminescence technique because of its high sensitivity, wide spectrum etc. in the field of veterinary drug residues have been paid close attention. This thesis has carried out the analysis of immune sulfonamides and Fluoroquinolones Residues in animal food bioluminescence, intended for animal drug residues Keep monitoring to provide rapid, sensitive and high-throughput detection methods. The expression of 1:1 fusion method respectively, alkaline phosphatase (Alkaline phosphatase, ALP) and Renilla luciferase (Renilla luciferase, Rluc) and fluoroquinolones (Fluoroquinolones, FQs) single chain antibody (single chain variable fragment, scFv) directional markers the FQs-scFv-ALP and FQs-scFv-Rluc fusion protein were established. The chemical can detect 20 kinds of luminescence of FQs direct competitive immunoassay method (CL-cdELISAFQs-scFv-ALP) and can detect 20 kinds of FQs bio Luminescent Immunoassay direct competition (BL-cdELIS AFQs-scFv-Rluc). With norfloxacin (Norfloxacin, NOR) respectively to establish the standard curve in CL-cdELISAFQs-scFv-ALP NOR IC50. The value is 0.15 g/L, the linear range is 0.04-1.08 ~ g/L, milk samples using ethyl acetate extraction, nitrogen blowing, the processing method of PBS complex solution After the elimination of matrix effect, the limit of detection (Limit of detection, LOD) for 0.021 g/L, between 0.43-133.89% and other cross reaction of 19 fluoroquinolones in BL-cdELISAFQs-scFv-Rluc NOR rate; IC50 value is 0.031 g/L, the linear range of 0.006-0.16 g/L, the milk sample by direct diluted 40 times can be eliminated matrix effects, LOD is 0.124 g/L, the rate between 0.45-129.7%. and other cross reaction of 19 fluoroquinolones with EU regulations of five fluoroquinolones in milk will be seized, ciprofloxacin (Ciprofloxacin, CIP), enrofloxacin (Enrofloxacin, ENR), marbofloxacin (Marbofloxacin, Danofloxacin, DAN (MAR) of danofloxacin flumequine) and (Flumequine, FLU) add recovery test, the CL-cdELISAFQs-scFv-ALP intra batch and inter batch recoveries were 78.2-119.4% and 79.6-117.4% respectively, the coefficient of variation 3.1-11.4 and 4.4-10.3%.BL-cdELISAFQs-scFv-Rluc in intra and inter batch recoveries were 78.4-117.2% and 77.8-117.9%, 6.9-13.4% and 7.4-12.9%. respectively. The coefficient of variation of the results showed that two kinds of the method of IC50 value than the traditional ELISA method were reduced by about 2 times and 10 times, IC50 bioluminescence value than Cl IC50 value low of about 5 times, and short time, the milk sample, while the coefficient of variation is greater than the bioluminescence chemiluminescence, but the recovery rate is 75-120%, the coefficient of variation is less than 15%, reached for the analysis of veterinary drug residues. Construction of sulfonamides (S ulfonamides, SAs) scFv and firefly luciferase (Firefly luciferase, Fluc) fusion protein (SAs-scFv-Fluc). FQs-scFv-Rluc and SAs-scFv-Fluc based on the simultaneous detection of 21 kinds of sulfonyl amine in milk and 20 kinds of fluoroquinolonic Nobel Quinolones residues in direct competition bioluminescence immunoassay (DBL-cdELISA). The NOR (Sulfamethazine, SMZ) and sulfamethazine respectively to establish the standard curve, NOR and SMZ IC50 were cross reaction of 0.051 g/L and 0.211 g/L. and 19 other fluoroquinolones ranged between 0.56-130.46%, between 2.25-1406.67%. milk diluted 80 times can eliminate the matrix effect and cross reaction of other 20 sulfonamides rate, NOR and SMZ LOD were 0.232 g/L and 1.894 g/L. were selected five fluoroquinolones (CIP, ENR, MAR, DAN and FLU) and five sulfonamides (SMZ, sulfadimethoxine (Sulfadimethoxine, SDM VA Lin), Sulfaquinoxaline (Sulfaquinoxaline, SQX), sulfamonomethoxine (Sulfamonomethoxine, SMM) and sulfamethoxazole (Sulfamethoxazole, SMX) of VA) to add five FQs recovery test. Add the recovery rate was 76.6-118.4%, the coefficient of variation was five SAs 5.7-9.7% recovery rate is 79.2-118.6%, the coefficient of variation is 5.4-9.6%, to meet the relevant requirements of veterinary drug residues detection. Using the dual luciferase substrate can be added at the same time, the signal collected at the same time, the double solid phase immune enzyme labeled detection and analysis of two kinds of material and method based on to Renilla luciferase and coelenterazine as energy donor, quantum dots as energy acceptor, fluoroquinolones as a model drug, a single chain antibody based on bioluminescence resonance energy transfer (QD-BRET) homogeneous immunoassay system. Renilla luciferase was the optimal substrate coelenterazine h QD-BRET, the Forster distance is 7.86nm and the distance between the donor and acceptor for 8.9nm. ENR to establish the standard curve, the IC50 value is 0.782 g/L, the linear range of 0.023-25.60 g/L, LOD 2.54 ng /L. and 7 generation The cross reaction of fluoroquinolones rate is 2.55-111.6%, consistent with the cross reaction rate. The third chapter second chapter five fluoroquinolones (CIP, ENR, MAR, DAN and FLU) intra and inter the recoveries were 78.5-118.2% and 79.3-116.6%, the coefficient of variation was less than 10%. while the sensitivity method less than DBL-cdELISA, but this method is a homogeneous analysis method, eliminating the coated, washing, short time, simple operation, and can meet the requirements of detection of residues of fluoroquinolones. In summary, this study established the homogeneous and single chain antibody and luciferase fusion protein based on heterogeneous biological immunoassay the analysis method can be used to detection of residues in milk of sulfonamides and fluoroquinolones, the maximum residue limits under the detection of 21 sulfonamides and 20 kinds of fluoroquinolones and. Chemiluminescence, bioluminescence, high sensitivity, short time, and the milk sample, combined application of different luciferase, can detect a variety of substances at the same time. In addition this study proved that quantum dots, QD-BRET system can be used for the detection of small molecule based, can be used as a veterinary drug residue monitoring in rapid, sensitive detection method high throughput.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S859.84
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本文编号：1667556