中国农业科学 ›› 2013, Vol. 46 ›› Issue (16): 3470-3477.doi: 10.3864/j.issn.0578-1752.2013.16.018

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

诺氟沙星ELISA试剂盒研制与性能鉴定

 邢广旭12, 王国栋3, 胡骁飞2, 王方雨2, 邓瑞广2, 张改平2   

  1. 1.河南农业大学牧医工程学院,郑州 450000
    2.河南省农业科学院免疫学重点实验室,郑州 450002
    3.安阳工学院,河南安阳 455000
  • 收稿日期:2013-03-13 出版日期:2013-08-15 发布日期:2013-06-25
  • 通讯作者: 通信作者张改平,Tel:0371-65711364;E-mail:zhanggaiping2003@163.com
  • 作者简介:邢广旭,Tel:0371-65738179;E-mail:xingguangxu@163.com
  • 基金资助:

    国家公益性行业(农业)科研专项(201203040);国家科技支撑计划(2011BAK10B00);河南省农科院521农业科技计划项目

Development and Identification of ELISA Kit for Norfloxacin Determination

 XING  Guang-Xu-12, WANG  Guo-Dong-3, HU  Xiao-Fei-2, WANG  Fang-Yu-2, DENG  Rui-Guang-2, ZHANG  Gai-Ping-2   

  1. 1.College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450000
    2. Key Lab for Animal Immunology, Henan Academy of Agricultural Science, Zhengzhou 450002
    3.Anyang Institute of Technology, Anyang 455000, Henan
  • Received:2013-03-13 Online:2013-08-15 Published:2013-06-25

摘要: 【目的】建立诺氟沙星(norfloxacin,Nor)免疫检测试剂盒。【方法】基于所制备的诺氟沙星单克隆抗体,通过矩阵法筛选最佳抗体工作浓度和二抗工作浓度,建立诺氟沙星间接竞争ELISA试剂盒(Nor-kit),并对其性能进行了鉴定和确证。【结果】Nor-kit的平均IC50 为5.18 μg•L-1,灵敏度为0.31 μg•L-1,检测限为1.00 μg•L-1。试剂盒除了和诺氟沙星反应外,还和同系物中的培氟沙星、氧氟沙星、环丙沙星、洛美沙星及恶喹酸有不同程度的交叉反应。奶样中回收率平均为103.23%,变异系数(CV)为9.33%。3个不同批次试剂盒的测定结果中,批内变异系数小于10%,且批间变异系数也小于10%;在4℃保存条件下,试剂盒可以保存6个月而质量不变。奶样添加回收试验,试剂盒与LC-MS/MS测定结果没有显著差异(P≥0.05);而且检测实际生产中鲜奶样时,试剂盒与LC-MS/MS测定结果也无显著差异(P≥0.05)。【结论】本研究成功研制出灵敏、特异、简便的诺氟沙星残留检测ELISA试剂盒。

关键词: 诺氟沙星 , 单克隆抗体 , ELISA , 试剂盒

Abstract: 【Objective】The objective of this experiment is to develop an ELISA kit for norfloxacin measurement. 【Method】Based on the prepared norfloxacin monoclonal antibody, the best work concentration of monoclonal antibody and HRP labeled antibody were selected by matrix method to develop an indirect competition ELISA kit for norfloxacin detection, and the properties of which also were examined and conformed. 【Result】The kit has average IC50 of 5.18 μg•L-1, a sensitivity of 0.31 μg•L-1, and the detection limit of 1.00 μg•L-1. The kit has different cross-reactivity with peifloxacin, ofloxacin, ciprofloxacin, lomefloxacin, oxilinic acid. The recoveries of norfloxacin in milk was 103.23%, and the CV was 9.33%. Among the detection results by three different batch kits, the inter-assay and intra-assay coefficient variation were all below 10%. The kit could remain effective for more than six months when stored at 4℃. There was no significant difference in the tested value of norfloxacin in milk between with NEO-kit and with LC-MS/ MS (P≥0.05). Furthermore, the tested value of norfloxacin in practice milk was no difference between with NEO-kit and with LC-MS/MS (P≥0.05). 【Conclusion】 A Nor-kit with high sensitivity, specificity and accuracy was developed in this experiment.

Key words: Norfloxacin , monoclonal antibody , ELISA , kit