基于免疫磁珠净化间接竞争酶联免疫吸附试验检测氟喹诺酮类药物  

Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Fluorquinolones based on Immunomagnetic Bead Purification

作　　者：黄婧洁 李苗 陈莹娴 钟雅兰 张婷婷[2] 姜廷超男 李建成[1,2] HUANG Jingjie;LI Miao;CHEN Yingxian;ZHONG Yalan;ZHANG Tingting;JIANG Tingchaonan;LI Jiancheng(Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety,China Agricultural University,Beijing 100193,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业大学,动物源性食品安全检测技术北京市重点实验室,北京100193 [2]中国农业大学动物医学院,北京100193

出　　处：《畜牧兽医学报》2023年第2期766-778,共13页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划(2018YFC1602301-4);国家自然科学基金(31972738)。

摘　　要：本研究旨在建立一种基于免疫磁珠进行分离、富集和净化前处理,检测鸡肉、鸡肝和鱼肉中氟喹诺酮类药物残留的间接竞争酶免疫吸附试验(indirect competitive enzyme-linked immunosorbent assay,icELISA)的研究方法。通过对合成免疫磁珠及免疫磁珠净化过程中单克隆抗体添加量、偶联时间、缓冲液pH、抗原添加量、抗原捕获时间、温度及包被条件等进行优化,初步建立了基于免疫磁珠净化的icELISA检测方法。结果显示:1)在1 mg的磁珠中,沙拉沙星(sarafloxacin,SAR)单克隆抗体最佳偶联量为15μg,偶联时间60 min,pH为4.4;2)最佳抗原添加量为1 ng·mL^(-1),捕获时间40 min,缓冲液为0.01 mol·L^(-1)PBS,IC50为0.73 ng·mL^(-1),线性范围为1.0~3.2 ng·mL^(-1);3)氟喹诺酮类药物在鸡肉、鸡肝、鱼肉的检测限均不超过1.33、2.17、2.31μg·kg^(-1),回收率为76.83%~98.70%,批内变异系数批间变异系数均不超过15%,经验证,鸡肉样本检测结果与高效液相色谱法(HPLC)结果一致。结果表明,与传统仪器检测方法相比,该方法提高了检测氟喹诺酮类药物的简便性、选择性以及检测效率,为氟喹诺酮类药物的残留检测提供了新思路。The aim of this study was to detect fluoroquinolones(FQs), in chicken, chicken liver, and fish, an indirect competitive enzyme-linked immunosorbent assay(icELISA) was developed based on immunomagnetic beads for sample clean-up and purification.(Method) Optimizing the addition amount of monoclonal antibody, coupling time, pH of buffer solution, the addition amount of quinolone antigen, capture time of antigen, temperature, and encapsulation condition of immunomagnetic beads, an icELISA method based on immunomagnetic beads clean-up was preliminarily established. Results were as follows: 1) In 1 mg magnetic beads, the optimal coupling amount of sarafloxacin(SAR) monoclonal antibody was 15 μg, coupling time was 60 min, pH was 4.4;2) The optimal antigen dosage was 1 ng·mL^(-1), capture time was 40 min, and the buffer ion concentration was 0.01 mol·L^(-1)PBS. The IC50was 0.73 ng·mL^(-1)with a linear range of 1.0-3.2 ng·mL^(-1);3) The detection limits of fluoroquinolones in chicken, chicken liver and fish were less than 1.33, 2.17 and 2.31 μg·kg^(-1), and the recoveries ranged from 76.83%-98.70% with intraday and interday variation coefficients lower than 15%. The recovery results were further confirmed using liquid chromatography-tandem mass spectrometry. The results suggest that, compared with the traditional instrumental detection method, this method can improve the simplicity, selectivity, and detection efficiency of fluoroquinolones, and provide a new idea for fluoroquinolones residue detection.

关 键 词：免疫磁珠富集 氟喹诺酮类药物 单克隆抗体 间接竞争酶联免疫吸附试验(icELISA) 

分 类 号：S859.84[农业科学—临床兽医学]