鸡组织中三种氟喹诺酮类药物残留检测的Ci-ELISA法及消除规律
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摘要
左氧氟沙星、环丙沙星、恩诺沙星属于第三代氟喹诺酮类药物,因为它们普遍具有抗菌谱广、抗菌活性强、消化吸收好与其他种类抗菌药物无交叉反应等特点,现已广泛大量地应用于畜禽养殖生产及临床用药上。由于其存在着潜在的毒副作用加上此类药物的大量及不合理使用,使得氟喹诺酮药物残留所带来的危害越来越受到关注,为了保障鸡肉产品的安全,需要建立一种高效、灵敏的药物残留检测技术。
本研究采用了免疫分析法中的间接竞争酶联免疫吸附测定法(Ci-ELISA),用左氧氟沙星、环丙沙星、恩诺沙星作为半抗原,制备出抗血清,分别建立Ci-ELISA去对鸡组织中氟喹诺酮药物残留进行检测并研究其消除规律。主要结果如下:
1.左氧氟沙星、恩诺沙星、环丙沙星人工抗原的合成鉴定及抗体的制备
用N-羟基琥珀酰亚胺法把左氧氟沙星(LVL)、恩诺沙星(ENR)、环丙沙星(CIF)、分别与载体蛋白牛血清白蛋白(BSA)偶联制备免疫抗原;用氯甲酸异丁酯法把左氧氟沙星(LVL)、恩诺沙星(ENR)、环丙沙星(CIF)分别与鸡卵清蛋白(OVA)偶联,制备包被检测抗原,采用紫外扫描法对合成的抗原进行鉴定,结果表明:免疫抗原和包被抗原均成功制备,结合比LVL-BSA、ENR-BSA、CIF-BSA、LVL-OVA、ENR-OVA、CIF-OVA,依次为10.6、11.6、13.6、5.1、5.2、6.8。
用三种人工免疫抗原LVL-BSA、ENR-BSA、CIF-BSA分别免疫家兔,制备出相应的抗血清,用ELISA法进行抗体效价检测,抗血清中的抗体效价均达到1:2~(12)以上,说明抗血清的制备是成功的。
2.三种氟喹诺酮类药物残留Ci-ELISA检测方法的建立
实验先对LVL、CIF、ENR进行交叉反应性的测定,测定结果表明,三种药物与氟喹诺酮类药物都具有一定的交叉反应,其中ENR与CIF的交叉反应最大。
通过对Ci-ELISA法实验条件的优化得到如下最佳反应参数:最适包被抗原工作浓度分别为1:200、1:200、1:400;抗LVL、ENR、CIF血清的最佳工作浓度为1:3200、1:6400、1:3200;最佳孵育时间为2h;酶标二抗(羊抗兔IgG-HRP)的稀释度为1:5000。
在上述优化的Ci-ELISA法反应参数的基础上,分别建立了LVL、ENR、CIF残留检测的Ci-ELISA法。具体结果如下:LVL检测的标准曲线的线性回归方程为Y=-0.2894X+0.0495(R~2=0.9878),中值(IC_(50))为64ng/mL,最低检测限(LOD)为10ng/mL,标准曲线的线性范围为10～1000ng/mL;ENR检测的标准曲线的线性回归方程为Y=-0.2186X+0.216(R~2=0.9843),中值(IC_(50))为36ng/mL,最低检测限(LOD)为10ng/mL,标准曲线的线性范围为10～1000ng/Ml;CIF检测的标准曲线的线性回归方程为Y=-0.219X+0.1661(R~2=0.9885),中值(IC_(50))为24ng/mL,最低检测限(LOD)为10ng/mL,标准曲线的线性范围为10～1000ng/mL。
通过添加回收试验,左氧氟沙星、环丙沙星、恩诺沙星药物在鸡肌肉、肝脏、肾脏组织中的平均添加回收率均在80%以上,批内和批间变异系数均低于15%,均达到了药物残留分析的要求。
3.Ci-ELISA检测方法研究鸡组织中三种氟喹诺酮类药物残留消除规律
本研究是以左氧氟沙星、环丙沙星和恩诺沙星3种FQs药物为对象,肉鸡按30mg/kg灌服给药,结果表明:3种FQs药物在停药96h后,在鸡组织中,肌肉中的药物浓度已经低于检测限,而肝脏、肾脏中仍可检测到;停药120h后,各组织中的药物浓度均低于检测限。
实验结果表明,本文用三种不同的氟喹诺酮类药物作为抗血清建立了Ci-ELISA检测氟喹诺酮类药物残留的方法,具有灵敏度高、精密度好、检测范围宽、最底检测限底的特点,达到了氟喹诺酮类药物残留分析的要求,具有一定的实践意义。
Levofloxacin,ciprofloxacin and enrofloxacin belong to the fluoroquinolones drug of the third generation,this kind of medicines generally have such characteristic as having wider antibacterial spectrum and stronger antibacterial activity,in addition that can be better absorbed by digest and have no cross reation with the other categeries of antibacterial drugs which now have been widely used as the clinical medication in the cultivation and production of the domestic animal and poulty. Because of this category of drugs,the hazard brought by the residue of the fluoroquinolones drug have been paid more and more attention to and to make sure of the product of chicken safety,the establishment of high performance and sensitive detective technology for the residual drugs is in need.This study use the method of immune analytical,and take levofloxacin,ciprofloxacin and enrofloxacin respectively as the half-antigen to prepare antiserum,then use the three drugs to establish the enzyme-linked immunosorbent assay of the indirect competitive of fluoroquinolones, as to find a method of sentitive and high performance to detective the residual drugs and to study their elimination.The result of experience as follows:
1.The synthesis indentity on the artificial antigen and preparation of antibody For levofloxacin,enrofloxaein and ciprofloxacin.
Take the levofloxacin,enrofloxacin and ciprofloxacin respectively to couple with the apoprotein to prepare the immunizing antigen by the N-hydroxide succinimide method;take the levofloxacin,enrofloxacin and ciprofloxacin respectively to couple with the OVA by the isobutyl chlonoformate method to prepare the peridiun detection antigen,make identifification on the synthetical antigen by the violet scanning method. The results indicate that immunizing antigen and coating antigen are all successful prepared.The conjugating ratios for LVL-BSA,ENR-BSA,CIF-BSA LVL-OVA,ENR-OVA,CIF-OVAis 10.6、11.6、13.6、5.1、5.2、6.8,respectively.
Use three kinds of artifical immunity antigen like LVL-BSA,ENR-BSA,CIF-BSA to immune the rabbit respectively to prepare the antiserum,make the antibody titer detection with ELISA method indirectly,if the antibody titer of the antiserum all are above 1:2~(12) and the preparation of antiserum is successful.
2.The establishment of enzyme-linked immunosorbent assay by indirect competition for three fluoroquinolones
Firstly,the experiment determinate the cross-reactive of LVL,CIF,ENR.The consequence indicate that the three kinds of drug all have unvarying cross-reaction with drugs of fluoroquinolones,and among the total the cross-raection between ENR and CIF have the best reflection.
Through the Ci-ELISA method optimized experimental conditions the best response to the following parameters.Coating antigen was optimal concentration of antigen were 1:200,1:200,1:400;Anti-LVL,ENR,CIF serum concentration of the best work 1:3200,1:6400,1:3200;and the best incubation time is 2h.The dilution of the enzyme symbolize to adope goat toresist rabbit IgG-HRP is 1:5000.
On the basis of the optimization of Ci-ELISA method reaction parameters.Use the serum of anti-LVL,anti-ENR,anti-CIF to establish the Ci-ELISA standard curve of LVL,ENR,CIF respectively,and we know that the equation of linear regression of the LVL standard curve is Y=-0.2894X+0.0495(R~2=0.9878),the midvalue(IC_(50)) is 64ng/ml,and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml;the equation of linear regression of the ENR standard curve is Y=-0.2186X+0.216(R~2=0.9843),the midvalue(IC_(50)) is 36ng/ml, and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml;the equation of linear regression of the CIF standard curve is Y= -0.219X+0.1661(R~2=0.9885),the midvalue(IC_(50)) is 24ng/ml,and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml.
By adding recovery rate test,make corresponding detect on the tissue of chicken (muscle,liver,kidney included),at the hand of recovery rate and variation coefficient, on average,the recovery rate of the kind of LVL,ENR,CIF drugs all are above 80%, the interassay and intrassay coefficient of variation are lowen then 15%,it seem that the recovery rate and variation coefficient of this experience both get to the requinement of the residual anylysis of the drugs.
3.Use Ci-ELISA detection method to study the residual drugs of three fluoroquinolones in the tissue of chicken and their elimination
The research take the LVL,ENR,CIF as the object,the edible chicken are drencked by 30mg/kg,the result shows that:96h after discontinuation,the drug concentration has been already lower then the limit of quantication in muscle but can be still found in liver and kidney;only more then 120h after discontinuation,the concentration of drug is lower then the limit of quantication in each tissue.
The result of the experiment indicate that the article use three kinds of drugs as the antiserum to establish the Ci-ELISA method to detect the residual drugs,which have the characteristic of high sensitivity and better degree of precision,wider detective range and lowest detectable limit,has achived the requirement of the residual drugs like fluoroquinolones also have determinate practical significance.
本研究采用了免疫分析法中的间接竞争酶联免疫吸附测定法(Ci-ELISA),用左氧氟沙星、环丙沙星、恩诺沙星作为半抗原,制备出抗血清,分别建立Ci-ELISA去对鸡组织中氟喹诺酮药物残留进行检测并研究其消除规律。主要结果如下:
1.左氧氟沙星、恩诺沙星、环丙沙星人工抗原的合成鉴定及抗体的制备
用N-羟基琥珀酰亚胺法把左氧氟沙星(LVL)、恩诺沙星(ENR)、环丙沙星(CIF)、分别与载体蛋白牛血清白蛋白(BSA)偶联制备免疫抗原;用氯甲酸异丁酯法把左氧氟沙星(LVL)、恩诺沙星(ENR)、环丙沙星(CIF)分别与鸡卵清蛋白(OVA)偶联,制备包被检测抗原,采用紫外扫描法对合成的抗原进行鉴定,结果表明:免疫抗原和包被抗原均成功制备,结合比LVL-BSA、ENR-BSA、CIF-BSA、LVL-OVA、ENR-OVA、CIF-OVA,依次为10.6、11.6、13.6、5.1、5.2、6.8。
用三种人工免疫抗原LVL-BSA、ENR-BSA、CIF-BSA分别免疫家兔,制备出相应的抗血清,用ELISA法进行抗体效价检测,抗血清中的抗体效价均达到1:2~(12)以上,说明抗血清的制备是成功的。
2.三种氟喹诺酮类药物残留Ci-ELISA检测方法的建立
实验先对LVL、CIF、ENR进行交叉反应性的测定,测定结果表明,三种药物与氟喹诺酮类药物都具有一定的交叉反应,其中ENR与CIF的交叉反应最大。
通过对Ci-ELISA法实验条件的优化得到如下最佳反应参数:最适包被抗原工作浓度分别为1:200、1:200、1:400;抗LVL、ENR、CIF血清的最佳工作浓度为1:3200、1:6400、1:3200;最佳孵育时间为2h;酶标二抗(羊抗兔IgG-HRP)的稀释度为1:5000。
在上述优化的Ci-ELISA法反应参数的基础上,分别建立了LVL、ENR、CIF残留检测的Ci-ELISA法。具体结果如下:LVL检测的标准曲线的线性回归方程为Y=-0.2894X+0.0495(R~2=0.9878),中值(IC_(50))为64ng/mL,最低检测限(LOD)为10ng/mL,标准曲线的线性范围为10～1000ng/mL;ENR检测的标准曲线的线性回归方程为Y=-0.2186X+0.216(R~2=0.9843),中值(IC_(50))为36ng/mL,最低检测限(LOD)为10ng/mL,标准曲线的线性范围为10～1000ng/Ml;CIF检测的标准曲线的线性回归方程为Y=-0.219X+0.1661(R~2=0.9885),中值(IC_(50))为24ng/mL,最低检测限(LOD)为10ng/mL,标准曲线的线性范围为10～1000ng/mL。
通过添加回收试验,左氧氟沙星、环丙沙星、恩诺沙星药物在鸡肌肉、肝脏、肾脏组织中的平均添加回收率均在80%以上,批内和批间变异系数均低于15%,均达到了药物残留分析的要求。
3.Ci-ELISA检测方法研究鸡组织中三种氟喹诺酮类药物残留消除规律
本研究是以左氧氟沙星、环丙沙星和恩诺沙星3种FQs药物为对象,肉鸡按30mg/kg灌服给药,结果表明:3种FQs药物在停药96h后,在鸡组织中,肌肉中的药物浓度已经低于检测限,而肝脏、肾脏中仍可检测到;停药120h后,各组织中的药物浓度均低于检测限。
实验结果表明,本文用三种不同的氟喹诺酮类药物作为抗血清建立了Ci-ELISA检测氟喹诺酮类药物残留的方法,具有灵敏度高、精密度好、检测范围宽、最底检测限底的特点,达到了氟喹诺酮类药物残留分析的要求,具有一定的实践意义。
Levofloxacin,ciprofloxacin and enrofloxacin belong to the fluoroquinolones drug of the third generation,this kind of medicines generally have such characteristic as having wider antibacterial spectrum and stronger antibacterial activity,in addition that can be better absorbed by digest and have no cross reation with the other categeries of antibacterial drugs which now have been widely used as the clinical medication in the cultivation and production of the domestic animal and poulty. Because of this category of drugs,the hazard brought by the residue of the fluoroquinolones drug have been paid more and more attention to and to make sure of the product of chicken safety,the establishment of high performance and sensitive detective technology for the residual drugs is in need.This study use the method of immune analytical,and take levofloxacin,ciprofloxacin and enrofloxacin respectively as the half-antigen to prepare antiserum,then use the three drugs to establish the enzyme-linked immunosorbent assay of the indirect competitive of fluoroquinolones, as to find a method of sentitive and high performance to detective the residual drugs and to study their elimination.The result of experience as follows:
1.The synthesis indentity on the artificial antigen and preparation of antibody For levofloxacin,enrofloxaein and ciprofloxacin.
Take the levofloxacin,enrofloxacin and ciprofloxacin respectively to couple with the apoprotein to prepare the immunizing antigen by the N-hydroxide succinimide method;take the levofloxacin,enrofloxacin and ciprofloxacin respectively to couple with the OVA by the isobutyl chlonoformate method to prepare the peridiun detection antigen,make identifification on the synthetical antigen by the violet scanning method. The results indicate that immunizing antigen and coating antigen are all successful prepared.The conjugating ratios for LVL-BSA,ENR-BSA,CIF-BSA LVL-OVA,ENR-OVA,CIF-OVAis 10.6、11.6、13.6、5.1、5.2、6.8,respectively.
Use three kinds of artifical immunity antigen like LVL-BSA,ENR-BSA,CIF-BSA to immune the rabbit respectively to prepare the antiserum,make the antibody titer detection with ELISA method indirectly,if the antibody titer of the antiserum all are above 1:2~(12) and the preparation of antiserum is successful.
2.The establishment of enzyme-linked immunosorbent assay by indirect competition for three fluoroquinolones
Firstly,the experiment determinate the cross-reactive of LVL,CIF,ENR.The consequence indicate that the three kinds of drug all have unvarying cross-reaction with drugs of fluoroquinolones,and among the total the cross-raection between ENR and CIF have the best reflection.
Through the Ci-ELISA method optimized experimental conditions the best response to the following parameters.Coating antigen was optimal concentration of antigen were 1:200,1:200,1:400;Anti-LVL,ENR,CIF serum concentration of the best work 1:3200,1:6400,1:3200;and the best incubation time is 2h.The dilution of the enzyme symbolize to adope goat toresist rabbit IgG-HRP is 1:5000.
On the basis of the optimization of Ci-ELISA method reaction parameters.Use the serum of anti-LVL,anti-ENR,anti-CIF to establish the Ci-ELISA standard curve of LVL,ENR,CIF respectively,and we know that the equation of linear regression of the LVL standard curve is Y=-0.2894X+0.0495(R~2=0.9878),the midvalue(IC_(50)) is 64ng/ml,and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml;the equation of linear regression of the ENR standard curve is Y=-0.2186X+0.216(R~2=0.9843),the midvalue(IC_(50)) is 36ng/ml, and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml;the equation of linear regression of the CIF standard curve is Y= -0.219X+0.1661(R~2=0.9885),the midvalue(IC_(50)) is 24ng/ml,and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml.
By adding recovery rate test,make corresponding detect on the tissue of chicken (muscle,liver,kidney included),at the hand of recovery rate and variation coefficient, on average,the recovery rate of the kind of LVL,ENR,CIF drugs all are above 80%, the interassay and intrassay coefficient of variation are lowen then 15%,it seem that the recovery rate and variation coefficient of this experience both get to the requinement of the residual anylysis of the drugs.
3.Use Ci-ELISA detection method to study the residual drugs of three fluoroquinolones in the tissue of chicken and their elimination
The research take the LVL,ENR,CIF as the object,the edible chicken are drencked by 30mg/kg,the result shows that:96h after discontinuation,the drug concentration has been already lower then the limit of quantication in muscle but can be still found in liver and kidney;only more then 120h after discontinuation,the concentration of drug is lower then the limit of quantication in each tissue.
The result of the experiment indicate that the article use three kinds of drugs as the antiserum to establish the Ci-ELISA method to detect the residual drugs,which have the characteristic of high sensitivity and better degree of precision,wider detective range and lowest detectable limit,has achived the requirement of the residual drugs like fluoroquinolones also have determinate practical significance.
引文
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