环丙沙星时间分辨荧光免疫分析方法的建立与应用

Development and Application of Time-resolved Fluroimmunoassay(TR-FIA) for Ciprofloxacin Determination

建立基于时间分辨荧光免疫分析技术的环丙沙星(CPF)间接竞争检测方法。将环丙沙星-卵清白蛋白(CPF-OVA)包被于固相微孔板,CPF标准品或样品中CPF与CPF包被原(CPF-OVA)共同竞争限量的CPF抗体,用铕标羊抗兔IgG示踪,建立环丙沙星时间分辨荧光免疫分析方法(CPF-TRFIA);考察包被质量浓度、抗体稀释液、反应时间等对方法灵敏度的影响。结果表明:在优化条件下,该方法的检测限为0.5μg/L,半抑制质量浓度(IC5 0)为4.32μg/L,线性范围(IC20～IC80)为1.14～28.85μg/L;该方法对蜂蜜中CPF平均回收率为80.66%～100.30%,对牛奶中CPF的平均回收率为76.30%～95.22%,两者的平均批内和批间变异系数分别为7.42%和10.92%。CPF-TRFIA间接竞争法测定环丙沙星具有灵敏度高、特异性好、性能稳定的优点,适于高通量筛查。

A time-resolved fluroimmunoassay（TRFIA） for the determination of ciprofloxacin（CPF） in an indirect competitive immunoassay mode was developed and applied.CPF-OVA was coated by physical adsorption onto a microtitre plate,and CPF standard or sample was competed with CPF-OVA for limited anti-CPF antibody,which was traced using europium-labeled goat anti rabbit IgG.The effects of experimental conditions including CPF-OVA concentration,buffer type,and reaction time on the sensitivity of TRFIA were explored.Under optimal conditions,the method revealed a detection limit of 0.5 μg/L,an IC50 of 4.32μg/L and a linear range（IC20-IC80） of 1.13-28.85 μg/L.The spike recovery rates of CPF in honey and milk at three spike levels were in the range of 80.66%-100.30% and 76.30%-95.22%,respectively,and the intra-batch and inter-batch coefficients of variation were 7.42% and 10.92%,respectively.The method was sensitive,specific,stable and suitable to be used for high-throughput determination of CPF residues.