用Ci-ELISA检测鸡组织中恩诺沙星残留
摘要
思诺沙星(ENR)属于氟喹诺酮类合成抗菌剂，用于治疗家畜禽感染性疾病。随着ENR的大量使用，残留于畜禽可食组织中的ENR及其代谢产物环丙沙星(CIP)引发了严重公共卫生问题。为此，欧盟规定在可食性动物组织中ENR和CIP的最高残留限量(MRL)为30μg／kg。目前，已建立了包括HPLC、ELISA在内的多种方法用于检测动物组织中的ENR残留。HPLC存在样品前处理严格，耗时，成本高等缺点；ELISA方法操作简单，经济而可行。
本研究旨在建立一种间接竞争酶联免疫吸附试验(Ci-ELISA)检测鸡组织中ENR残留。通过N-羟基琥珀酰亚胺活性酯法(NHS法)将半抗原ENR分别与蛋白质载体牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联成为全抗原ENR-BSA和ENR-OVA。ENR-BSA作为免疫原免疫家兔制备抗血清，ENR-OVA在ELISA中作为包被原。用方阵滴定法确定包被原(ENR-OVA)、抗血清、羊抗兔IgG-HRP的最佳工作浓度及反应时间，而后在此基础上建立Ci-ELISA方法检测实验肉仔鸡组织中ENR残留情况，并对方法的精密度、准确度、灵敏度和选择性指标进行评价。其结果如下：
1．ENR-BSA，ENR-OVA，ENR，BSA和OVA五种溶液经过200nm～400nm波长紫外扫描，BSA和OVA分别在289nm和291nm波长处有最大吸收峰，ENR在333nm波长处有最大吸收峰，ENR-BSA和ENR-OVA的最大吸收峰分别在319nm和320nm波长处，此外还分别都在332nm波长处有一吸收峰，说明ENR-BSA和ENR-OVA的吸收光谱是ENR的吸收光谱与蛋白质吸收光谱的整和，其中含有ENR特征峰。该结果表明ENR已与蛋白质载体成功偶联。
2．用间接ELISA法检测制得的抗血清效价在1：3200～1：6400，最佳工作浓度(OD值=0.4)为1：800，包被原ENR-OVA的最佳工作浓度为1：40，羊抗兔IgG-HRP的稀释浓度为1：1000。
3．建立的检测ENR的Ci-ELISA方法在PBS介质中ENR不同浓度(3000，300，30，3，0.3，0.03ng／ml)的情况下表示其精密度的批内变异系数和批间变异系数分别为4.5～38.1％和8.8～41.9％；表示方法准确度的鸡肌肉、肝脏和肾脏中ENR添加平均回收率分别为35.48～63.74％，69.08～86.97％和51.04～72.59％；表示方法灵敏度的ENR在鸡肌肉、肝脏、肾脏和PBS中的最低检测限(LOD)分别为7.82，12.71，8.75和1.62ng／ml；在PBS中ENR 50％抑制浓度(I_(50))为16.26ng／ml，环丙沙星、氧氟沙星、庆大霉素、青霉素钾和磺胺嘧啶五种药物的I_(50)均高于3000ng／ml，它们与ENR的交叉反应率均低于0.54％，即抗血清与其它氟喹诺酮类药物和
摘要
目巨绝里互旦目里口口级组口旦亘旦巨旦旦口国旦国纽里皿里旦
抗生素均无交叉反应性。ENR在鸡肌肉、肝脏和肾脏中的150分别为63.97，45.92和50.47ng
/ml。ENR在肌肉、肝脏、肾脏和PBS中的标准回归方程式分别为Y二76.720一14.792X(r=一0.97
6，n=16)，Y=75.807一15.525X(r=一0.981，n=16)，Y=74.631一14.461X(r=一0.992，n=16)和Y=68.
006一14.865x(r二一0.997，下16)，ENR在鸡肌肉、肝脏、肾脏和PBS介质中标准曲线的线性范
围分别为7.82~3000ng/ml，12.71~3000ng/ml，8.75~3000n岁ml和1.62~3000ng/ml。以
上结果说明该方法特异性好，选择性高，灵敏度、精密度和准确度均基本符合残留检测要求。
4.由本试验建立的Ci一ELISA检测鸡组织中ENR残留，结果为休药第2天，鸡组织(肌肉、肝
脏和肾脏)中ENR的残留量为30~300ng/g，而且肝脏和肾脏的残留量均高于肌肉;休药第6
天，肌肉、肝脏和肾脏的残留量都在3~3Ong/g之间;休药第10和14天，检测结果表明肌
肉、肝脏和肾脏中ENR残留量都在3ng/g以下。说明给肉仔鸡连续4天口服10mg/kg体重剂
量的ENR上市前至少需休药6天，以避免该类抗菌药在肉仔鸡体内的残留。
研究表明，本实验室建立的检测鸡组织中ENR残留的Ci一ELISA是一种特异性良好，简便
且价廉的方法。
Enrofloxacin(ENR) is a synthetic antibacterial agent that belongs to the fluoroquinolone group. It has been increasingly used in veterinary medicine to treat microbial infections, however, the ENR residues in animal edible tissues could cause serious public health problems. An European Commission Regulation has set a maximum residue limit(MRL) of 30ug/kg for the sum of ENR and its metabolite ciprofloxacin(CIP) in animal edible tissues. So far, several methods have been presented for the detection of ENR in animal edible tissues, including high performance liquid chromatography(HPLC) and enzyme-linked immnosorbent assays(ELISA).HPLC is time-consuming, expensive, labor-intensive and requires extensive sample clean-up. ELISA is convenient to perform, economical and reliable.
The purpose of the study here is to develop an indirect competitive enzyme-linked immunosorbent assays(Ci-ELISA) to monitor ENR in chicken tissues. Bovine serum albumin(BSA) and ovalbumin(OVA) were used as protein carriers respectively to couple with hapten ENR by an active ester method, and complete antigens ENR-BSA and ENR-OVA were prepared. ENR-BSA acted as immunogen which immunized rabbits to produce antisera, while ENR-OVA acted as coating antigen in ELISA. The optimal concentration of coating antigen(ENR-OVA), antisera, horseradish peroxidase coupled to the goat IgG of anti-rabbit IgG and the optimal reaction time were determined by the phalanx titrimetric analysis. Thereafter, an Ci-ELISA was established to detect ENR residues in the experimental broiler chickens tissues and was evaluated precision, accuracy, sensitivity and selectivity of the method. The results were as followed:
1.The solutions of ENR-BSA, ENR-OVA, ENR, BSA and OVA were scanned with ultraviolet ray absorbance detection at the range of 200nm~400nm, there was a maximum absorbance peak at 289, 291,333,319 and 320nm wavelength of spectrum of BSA, OVA, ENR, ENR-BSA and ENR-OVA, respectively. In addition, the spectrum of ENR-BSA and ENR-OVA both had an absorbance peak at 332nm wavelength. Their spectrum showed the fact that the spectrum of ENR-BSA and ENR-OVA are the integration of the spectrum of ENR and proteins, including ENR characteristic absorbance peak. The result suggested that it is successful to link between ENR and protein carriers.
2.The antiserum litre obtained using an indirect ELISA was at the range of 1:3200 to 1:6400, its optimal working concentration was 1:800, the optimal concentration of coating antigen(ENR-OVA) and horseradish peroxidase marked to the goat IgG of anti-rabbit IgG were 1:40 and 1:1000. 3.Precision of the Ci-ELlSA employed to detect ENR indicated by coefficients of variation of intra-assay and inter-assay were 4.5~38.1% and 8.8~41.9% respectively, according to ENR different concentrations(3000, 300, 30, 3, 0.3, 0.03ng/ml) in PBS. Accuracy of the method denoted by mean recoveries of ENR at levels of 3000, 300, 30, 3, 0.3, 0.03ng/ml in spiked chicken muscle, liver and kidney samples were 35.48~63.74%, 69.08~86.97% and 51.04~72.59%, respectively. Sensitivity of the method determined by limit of detection(LOD) in muscle, liver, kidney and PBS were 7.82, 12.71, 8.75 and 1.62ng/ml, respectively. ENR concentration of 50% ENR inhibited (I50) in PBS was 16.26ng/ml, I50 of ciprofloxacin, ofloxacin, gentamicin, benzylpenicillin potassium and sulfadiazine were all over 3000ng/ml, cross-reaction between those five antimicrobial agents and ENR were all below 0.54%, that is to say, the antisera had no cross-reactivity with other fluoroquinolones and antibiotics. I50 of ENR in chicken muscle, liver and kidney samples were 63.97, 45.92 and 50.47ng/ml, respectively. The standard regression equation was Y=76.720-14.792X(r=-0.976,n=16) in muscle, Y=75.807-15.525X(r=-0.981,n=16) in liver, Y=74.631-14.461X(r=-0.992,n=16) in kidney and Y=68.006-14.865X(r=-0.997,n=16) in PBS, linear range was 7.82~3000ng/ml in muscle, 12.71~3000ng/ml in liver, 8.75~3000ng/ml in kidney and 1.62~3000ng/ml in PBS. These results mentioned above showed the method has good specifi
本研究旨在建立一种间接竞争酶联免疫吸附试验(Ci-ELISA)检测鸡组织中ENR残留。通过N-羟基琥珀酰亚胺活性酯法(NHS法)将半抗原ENR分别与蛋白质载体牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联成为全抗原ENR-BSA和ENR-OVA。ENR-BSA作为免疫原免疫家兔制备抗血清，ENR-OVA在ELISA中作为包被原。用方阵滴定法确定包被原(ENR-OVA)、抗血清、羊抗兔IgG-HRP的最佳工作浓度及反应时间，而后在此基础上建立Ci-ELISA方法检测实验肉仔鸡组织中ENR残留情况，并对方法的精密度、准确度、灵敏度和选择性指标进行评价。其结果如下：
1．ENR-BSA，ENR-OVA，ENR，BSA和OVA五种溶液经过200nm～400nm波长紫外扫描，BSA和OVA分别在289nm和291nm波长处有最大吸收峰，ENR在333nm波长处有最大吸收峰，ENR-BSA和ENR-OVA的最大吸收峰分别在319nm和320nm波长处，此外还分别都在332nm波长处有一吸收峰，说明ENR-BSA和ENR-OVA的吸收光谱是ENR的吸收光谱与蛋白质吸收光谱的整和，其中含有ENR特征峰。该结果表明ENR已与蛋白质载体成功偶联。
2．用间接ELISA法检测制得的抗血清效价在1：3200～1：6400，最佳工作浓度(OD值=0.4)为1：800，包被原ENR-OVA的最佳工作浓度为1：40，羊抗兔IgG-HRP的稀释浓度为1：1000。
3．建立的检测ENR的Ci-ELISA方法在PBS介质中ENR不同浓度(3000，300，30，3，0.3，0.03ng／ml)的情况下表示其精密度的批内变异系数和批间变异系数分别为4.5～38.1％和8.8～41.9％；表示方法准确度的鸡肌肉、肝脏和肾脏中ENR添加平均回收率分别为35.48～63.74％，69.08～86.97％和51.04～72.59％；表示方法灵敏度的ENR在鸡肌肉、肝脏、肾脏和PBS中的最低检测限(LOD)分别为7.82，12.71，8.75和1.62ng／ml；在PBS中ENR 50％抑制浓度(I_(50))为16.26ng／ml，环丙沙星、氧氟沙星、庆大霉素、青霉素钾和磺胺嘧啶五种药物的I_(50)均高于3000ng／ml，它们与ENR的交叉反应率均低于0.54％，即抗血清与其它氟喹诺酮类药物和
摘要
目巨绝里互旦目里口口级组口旦亘旦巨旦旦口国旦国纽里皿里旦
抗生素均无交叉反应性。ENR在鸡肌肉、肝脏和肾脏中的150分别为63.97，45.92和50.47ng
/ml。ENR在肌肉、肝脏、肾脏和PBS中的标准回归方程式分别为Y二76.720一14.792X(r=一0.97
6，n=16)，Y=75.807一15.525X(r=一0.981，n=16)，Y=74.631一14.461X(r=一0.992，n=16)和Y=68.
006一14.865x(r二一0.997，下16)，ENR在鸡肌肉、肝脏、肾脏和PBS介质中标准曲线的线性范
围分别为7.82~3000ng/ml，12.71~3000ng/ml，8.75~3000n岁ml和1.62~3000ng/ml。以
上结果说明该方法特异性好，选择性高，灵敏度、精密度和准确度均基本符合残留检测要求。
4.由本试验建立的Ci一ELISA检测鸡组织中ENR残留，结果为休药第2天，鸡组织(肌肉、肝
脏和肾脏)中ENR的残留量为30~300ng/g，而且肝脏和肾脏的残留量均高于肌肉;休药第6
天，肌肉、肝脏和肾脏的残留量都在3~3Ong/g之间;休药第10和14天，检测结果表明肌
肉、肝脏和肾脏中ENR残留量都在3ng/g以下。说明给肉仔鸡连续4天口服10mg/kg体重剂
量的ENR上市前至少需休药6天，以避免该类抗菌药在肉仔鸡体内的残留。
研究表明，本实验室建立的检测鸡组织中ENR残留的Ci一ELISA是一种特异性良好，简便
且价廉的方法。
Enrofloxacin(ENR) is a synthetic antibacterial agent that belongs to the fluoroquinolone group. It has been increasingly used in veterinary medicine to treat microbial infections, however, the ENR residues in animal edible tissues could cause serious public health problems. An European Commission Regulation has set a maximum residue limit(MRL) of 30ug/kg for the sum of ENR and its metabolite ciprofloxacin(CIP) in animal edible tissues. So far, several methods have been presented for the detection of ENR in animal edible tissues, including high performance liquid chromatography(HPLC) and enzyme-linked immnosorbent assays(ELISA).HPLC is time-consuming, expensive, labor-intensive and requires extensive sample clean-up. ELISA is convenient to perform, economical and reliable.
The purpose of the study here is to develop an indirect competitive enzyme-linked immunosorbent assays(Ci-ELISA) to monitor ENR in chicken tissues. Bovine serum albumin(BSA) and ovalbumin(OVA) were used as protein carriers respectively to couple with hapten ENR by an active ester method, and complete antigens ENR-BSA and ENR-OVA were prepared. ENR-BSA acted as immunogen which immunized rabbits to produce antisera, while ENR-OVA acted as coating antigen in ELISA. The optimal concentration of coating antigen(ENR-OVA), antisera, horseradish peroxidase coupled to the goat IgG of anti-rabbit IgG and the optimal reaction time were determined by the phalanx titrimetric analysis. Thereafter, an Ci-ELISA was established to detect ENR residues in the experimental broiler chickens tissues and was evaluated precision, accuracy, sensitivity and selectivity of the method. The results were as followed:
1.The solutions of ENR-BSA, ENR-OVA, ENR, BSA and OVA were scanned with ultraviolet ray absorbance detection at the range of 200nm~400nm, there was a maximum absorbance peak at 289, 291,333,319 and 320nm wavelength of spectrum of BSA, OVA, ENR, ENR-BSA and ENR-OVA, respectively. In addition, the spectrum of ENR-BSA and ENR-OVA both had an absorbance peak at 332nm wavelength. Their spectrum showed the fact that the spectrum of ENR-BSA and ENR-OVA are the integration of the spectrum of ENR and proteins, including ENR characteristic absorbance peak. The result suggested that it is successful to link between ENR and protein carriers.
2.The antiserum litre obtained using an indirect ELISA was at the range of 1:3200 to 1:6400, its optimal working concentration was 1:800, the optimal concentration of coating antigen(ENR-OVA) and horseradish peroxidase marked to the goat IgG of anti-rabbit IgG were 1:40 and 1:1000. 3.Precision of the Ci-ELlSA employed to detect ENR indicated by coefficients of variation of intra-assay and inter-assay were 4.5~38.1% and 8.8~41.9% respectively, according to ENR different concentrations(3000, 300, 30, 3, 0.3, 0.03ng/ml) in PBS. Accuracy of the method denoted by mean recoveries of ENR at levels of 3000, 300, 30, 3, 0.3, 0.03ng/ml in spiked chicken muscle, liver and kidney samples were 35.48~63.74%, 69.08~86.97% and 51.04~72.59%, respectively. Sensitivity of the method determined by limit of detection(LOD) in muscle, liver, kidney and PBS were 7.82, 12.71, 8.75 and 1.62ng/ml, respectively. ENR concentration of 50% ENR inhibited (I50) in PBS was 16.26ng/ml, I50 of ciprofloxacin, ofloxacin, gentamicin, benzylpenicillin potassium and sulfadiazine were all over 3000ng/ml, cross-reaction between those five antimicrobial agents and ENR were all below 0.54%, that is to say, the antisera had no cross-reactivity with other fluoroquinolones and antibiotics. I50 of ENR in chicken muscle, liver and kidney samples were 63.97, 45.92 and 50.47ng/ml, respectively. The standard regression equation was Y=76.720-14.792X(r=-0.976,n=16) in muscle, Y=75.807-15.525X(r=-0.981,n=16) in liver, Y=74.631-14.461X(r=-0.992,n=16) in kidney and Y=68.006-14.865X(r=-0.997,n=16) in PBS, linear range was 7.82~3000ng/ml in muscle, 12.71~3000ng/ml in liver, 8.75~3000ng/ml in kidney and 1.62~3000ng/ml in PBS. These results mentioned above showed the method has good specifi
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