氟喹诺酮类药物多残留间接竞争ELISA检测方法的建立

Development of indirect competitive ELISA for detection of fluoroquinolone residues

为建立同时检测多种氟喹诺酮药物的免疫学分析方法,本研究以碳二亚胺(EDC)二步法合成环丙沙星人工抗原,免疫新西兰大白兔制备多克隆抗体,建立氟喹诺酮类药物多残留检测方法。标准曲线在PBS中的线性检测范围为0.2 ng/mL～376 ng/mL,半数抑制浓度(IC50)为8 ng/mL,检测限(LOD)为0.1 ng/mL;抗体对恩诺沙星、诺氟沙星和培氟沙星均能特异性识别,IC50值分别为7.3 ng/mL、10.6 ng/mL、和13.2 ng/mL;标准品稀释液中NaOH和甲醇的最高容忍度分别为10%和30%;牛奶基质中添加5 ng/mL、20 ng/mL和50 ng/mL的标准品,环丙沙星、恩诺沙星、诺氟沙星和培氟沙星的回收率分别为93%～108%、96%～110%、92.5%～104%和93%～102%。

To develop a multidetermination immunoassay for fluoroquinolone（FQs） residues,an indirect competitive ELISA was developed with the polyclonal antibody against ciprofloxacin（CPFX pAb） produced from rabbits which immunized with FQs conjugated BSA.This method was sensitive and had a linear detection range from 0.2 ng/mL to 376 ng/mL（R2=0.943）,with IC50 and LOD values of 8 ng/mL and 0.1 ng/mL.The antibody exhibited high sensitivity to FQs,and the IC50 values to enrofloxacin,norfloxacin and pefloxacin were 7.3 ng/mL,10.6 ng/mL,and 13.2 ng/mL,respectively.The optimized detecting condition indicated that the concentrations of NaOH and methanol in dilute solution was less than 10% and 30%.When the ciprofloxacin,enrofloxacin,norfloxacin and pefloxacin in milk were at highest concentrations of 5 ng/mL,20 ng/mL and 50 ng/mL,the recoveries were ranged from 93% to 108%,96% to 110%,92.5% to 104%,and 93% to 102%,respectively.