伏安免疫法检测牛奶中氯霉素残留

为探索用于现场检测牛乳中氯霉素(CAP)残留的高灵敏度及特异性强的免疫传感器方法,本实验在制备CAP单克隆抗体的基础上,以卵清蛋白-氯霉素(OVA-CAP)偶联物为包被抗原,并将其包被到聚苯乙烯反应板上;在孵育反应中,样品中的CAP与OVA-CAP竞争结合CAP单克隆抗体,洗涤后加入碱性磷酸酶(ALP)标记的二抗,经再次孵育及洗涤后加入对硝基苯磷酸(pNPP)底物液;反应终止后用线性导数伏安法记录pNPP水解产物的氧化峰电流。实验结果表明,用免疫传感法测试CAP的灵敏度高于传统的间接竞争酶联免疫吸附法(ELISA)。该方法检测CAP的检出限为0.064μg/L;检测线性范围为0.15~600μg/L,测试牛奶样品的平均回收率为89.8%。另外,由于免疫电化学传感器体积较小,便于携带,操作简单,可实现牛乳样品中CAP残留的现场检测。

Immuno-voltammetric Determination of Chloramphenicol Residues in Milk

To investigate the immunovoltammetric techniques with high sensitivity and specificity for the determination of chloramphenicol (CAP) residues in milk, the conjugate of ovalbumin (OVA)-CAP was immobilized onto the micro-reaction plate and the monoclonal anti-chloramphenicol antibody was prepared to set up competitive enzyme-linked immunoabsorbent assays (ELISA). After the procedure of competitive combination of OVA-CAP and CAP in sample with anti-chloramphenicol monoclonal antibody during the incubation period, the plate was rinsed and alkaline phosphatase (ALP)-labeled goat anti-mouse IgG was added into the microcuvettes. p-nitrophenylphate was applied as substrate after the second incubation and then rinsed. The oxidative peak currents (OPC) were recorded by differentiated pulse voltammetry (DPV) after the reaction on the plate, which was terminated by the addition of 2 M NaOH. The results showed that the sensitivity of immunovoltammetry was better than that of ELISA for determination of CAP residues. This method allows the detection limit as low as 0.064 μg l⁻¹ with the linear range of 0.15–600 μg l⁻¹ CAP, and the average recovery reaches 89.8% based on the milk sample. Furthermore, the immunovoltammetric apparatus is portable and can be used for the on-spot determination of CAP residues in milk due to its small size and easy operation.