金刚烷胺残留化学发光酶免疫法的建立
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摘要
建立金刚烷胺残留的化学发光酶免疫方法。对酶标抗原与磁标抗体的最佳稀释倍数、反应时间、底物发光时间进行优化,建立标准曲线,同时对方法的检测限、准确度和精密度进行评价。结果表明:50%抑制浓度(IC50)为0.481μg/L,线性范围是0.085～2.729μg/L;在动物组织、兽药、饲料中的检测限为0.1μg/kg,样本添加回收率为80.2%～94.5%,批内、批间相对标准偏差均<10%。该方法灵敏度高、操作简便,可广泛用于动物组织、兽药、饲料中金刚烷胺残留量的测定。
The aim of the study is to establish a method for determination of amantadine residues by chemiluminescent enzyme immunoassay( CLEIA). After optimizing the dilution multiple of enzyme-labeled antigen and magnetic labeled antibody,reaction time and substrate luminescence time,a standard curve was established,and finally the detection limit,accuracy and precision of the method were evaluated.The results showed that the 50% inhibitory concentration( IC_(50)) was 0.481 μg/L,and the linearity range was 0.085～2.729 μg/L.The detection limit in animal tissues,veterinary drugs and feed were 0.1 μg/kg.The recoveries ranged from 80.2% ～94.5%,with the intra-assay and inter-assay coefficients of variation less than 10%.The method is sensitive,simple and it can be widely used for the determination of amantadine residues in animal tissues,veterinary drugs and feed.
The aim of the study is to establish a method for determination of amantadine residues by chemiluminescent enzyme immunoassay( CLEIA). After optimizing the dilution multiple of enzyme-labeled antigen and magnetic labeled antibody,reaction time and substrate luminescence time,a standard curve was established,and finally the detection limit,accuracy and precision of the method were evaluated.The results showed that the 50% inhibitory concentration( IC_(50)) was 0.481 μg/L,and the linearity range was 0.085～2.729 μg/L.The detection limit in animal tissues,veterinary drugs and feed were 0.1 μg/kg.The recoveries ranged from 80.2% ～94.5%,with the intra-assay and inter-assay coefficients of variation less than 10%.The method is sensitive,simple and it can be widely used for the determination of amantadine residues in animal tissues,veterinary drugs and feed.
引文
[1]胡巧茹,周长朋,张玉春,等.高效液相色谱-串联质谱法测定辣椒及辣椒制品中金刚烷胺残留[J].分析测试学报,2014,33(10):1184-1188.
[2]赵桂宏,白向荣,李晓玲,等.金刚烷胺治疗帕金森病的有效性和安全性评价[J].中国药学杂志,2011,46(23):1847-1850.
[3]云环,崔凤云,严华,等.超高效液相色谱-串联质谱法测定鸡肉中的利巴韦林和金刚烷胺[J].色谱,2013,31(8):724-728.
[4]Yavarian J,Azad T M,Zheng X,et al.Amantadine resistance in relation to the evolution of influenza A(H3N2)viruses in Iran[J].Antiviral Research,2010,88(2):193-196.
[5]刘华雷,王志亮,吕艳,等.不同亚型禽流感病毒对金刚烷胺的耐药性分析[C].中国畜牧兽医学会兽医公共卫生学分会第二次学术研讨会论文集,2010.
[6]吴松松.金刚烷胺免疫学快速定量检测方法初步研究[D].南昌:南昌大学,2016.
[7]中华人民共和国农业部公告(第560号)[J].中华人民共和国农业部公报,2005,10(11):40.
[8]Farajzadeh M A,Nouri N,Nabil A A A.Determination of amantadine in biological fluids using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection[J].Journal of Chromatography B Analytical Technologies in the Biomedical&Life Sciences,2013,940(4):142-149.
[9]Suo D C,Wang P L,Li Y,et al.Simultaneous determination of antibiotics and amantadines in animal-derived feedstuffs by ultraperformance liquid chromatographic-tandem mass spectrometry[J].Journal of Chromatography B,2018,1095:183-190.
[10]苏娟.高灵敏金刚烷胺胶体金免疫试纸条的研制[D].杭州:浙江工业大学,2015.
[11]吴小平,王文珺,刘萤,等.金刚烷胺ELISA试剂盒的研制[J].检验检疫学刊,2015,25(4):68-70.
[12]叶云锋,李研东,吴雨洋,等.化学发光免疫分析方法检测粮食谷物中赭曲霉毒素A残留[J].食品安全质量检测学报,2017,8(7):2787-2791.
[13]何方洋.兽药残留检测技术研究[J].食品科学技术学报,2012,30(1):17-19.
[14]罗立梅,张彬,陈刚.化学发光微粒子免疫分析法检测性激素6项的性能验证[J].国际检验医学杂志,2017,38(9):1214-1216.
[15]Luo P J,Zhang J B,Wang H L,et al.Rapid and sensitive chemiluminescent enzyme immunoassay for the determination of neomycin residues in milk[J].Biomedical and Environmental Sciences,2016,29(5):374-378.
[16]冯才伟,黄健,王兆芹,等.检测动物组织中金刚烷胺残留的酶联免疫试剂盒的研制[J].畜牧与兽医,2016,48(11):27-31.
[17]戴尽波,徐振林,刘凤银,等.化学发光酶免疫分析测定鱼肉中呋喃它酮代谢物方法研究[J].分析化学,2015,43(6):871-875.
[18]董李学,冯才伟,冯静,等.抗氧氟沙星单克隆抗体的制备及ELISA快速试剂盒的初步研制[J].中国畜牧兽医,2014,41(8):90-94.
[19]张荣升.脱氧雪腐镰刀菌烯醇单克隆抗体的制备及化学发光酶免疫分析方法的建立[D].南京:南京农业大学,2015.
[20]丁丽华,于斐,张冠军,等.脱氧雪腐镰刀菌烯醇的高灵敏化学发光磁酶免疫法检测[J].郑州大学学报(医学版),2017,52(2):134-137.
[2]赵桂宏,白向荣,李晓玲,等.金刚烷胺治疗帕金森病的有效性和安全性评价[J].中国药学杂志,2011,46(23):1847-1850.
[3]云环,崔凤云,严华,等.超高效液相色谱-串联质谱法测定鸡肉中的利巴韦林和金刚烷胺[J].色谱,2013,31(8):724-728.
[4]Yavarian J,Azad T M,Zheng X,et al.Amantadine resistance in relation to the evolution of influenza A(H3N2)viruses in Iran[J].Antiviral Research,2010,88(2):193-196.
[5]刘华雷,王志亮,吕艳,等.不同亚型禽流感病毒对金刚烷胺的耐药性分析[C].中国畜牧兽医学会兽医公共卫生学分会第二次学术研讨会论文集,2010.
[6]吴松松.金刚烷胺免疫学快速定量检测方法初步研究[D].南昌:南昌大学,2016.
[7]中华人民共和国农业部公告(第560号)[J].中华人民共和国农业部公报,2005,10(11):40.
[8]Farajzadeh M A,Nouri N,Nabil A A A.Determination of amantadine in biological fluids using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection[J].Journal of Chromatography B Analytical Technologies in the Biomedical&Life Sciences,2013,940(4):142-149.
[9]Suo D C,Wang P L,Li Y,et al.Simultaneous determination of antibiotics and amantadines in animal-derived feedstuffs by ultraperformance liquid chromatographic-tandem mass spectrometry[J].Journal of Chromatography B,2018,1095:183-190.
[10]苏娟.高灵敏金刚烷胺胶体金免疫试纸条的研制[D].杭州:浙江工业大学,2015.
[11]吴小平,王文珺,刘萤,等.金刚烷胺ELISA试剂盒的研制[J].检验检疫学刊,2015,25(4):68-70.
[12]叶云锋,李研东,吴雨洋,等.化学发光免疫分析方法检测粮食谷物中赭曲霉毒素A残留[J].食品安全质量检测学报,2017,8(7):2787-2791.
[13]何方洋.兽药残留检测技术研究[J].食品科学技术学报,2012,30(1):17-19.
[14]罗立梅,张彬,陈刚.化学发光微粒子免疫分析法检测性激素6项的性能验证[J].国际检验医学杂志,2017,38(9):1214-1216.
[15]Luo P J,Zhang J B,Wang H L,et al.Rapid and sensitive chemiluminescent enzyme immunoassay for the determination of neomycin residues in milk[J].Biomedical and Environmental Sciences,2016,29(5):374-378.
[16]冯才伟,黄健,王兆芹,等.检测动物组织中金刚烷胺残留的酶联免疫试剂盒的研制[J].畜牧与兽医,2016,48(11):27-31.
[17]戴尽波,徐振林,刘凤银,等.化学发光酶免疫分析测定鱼肉中呋喃它酮代谢物方法研究[J].分析化学,2015,43(6):871-875.
[18]董李学,冯才伟,冯静,等.抗氧氟沙星单克隆抗体的制备及ELISA快速试剂盒的初步研制[J].中国畜牧兽医,2014,41(8):90-94.
[19]张荣升.脱氧雪腐镰刀菌烯醇单克隆抗体的制备及化学发光酶免疫分析方法的建立[D].南京:南京农业大学,2015.
[20]丁丽华,于斐,张冠军,等.脱氧雪腐镰刀菌烯醇的高灵敏化学发光磁酶免疫法检测[J].郑州大学学报(医学版),2017,52(2):134-137.