诺氟沙星免疫学检测方法的建立及优化

Development and Optimization of Immunoassay Method for Detection of Norfloxacin Residues

采用优化的碳二亚胺法制备诺氟沙星(NOR)人工抗原,免疫新西兰大白兔制备多克隆抗体,建立间接竞争ELISA标准曲线。对NOR的线性检测范围为0.07～31.8ng/mL,半数抑制质量浓度(IC50)和最低检测限(LOD)分别为1.5ng/mL和0.04ng/mL。检测缓冲液中pH值和磷酸根离子浓度的最佳参数分别为7.4和10mmol/L;对甲醇和NaOH溶液(30mmol/L)的最佳调整体积分数分别为30%和10%。抗体具有广谱特异性,与环丙沙星(83.3%)、培氟沙星(78.9%)、依诺沙星(68.2%)、恩诺沙星(51.7%)和洛美沙星(41.7%)有较高的交叉反应率,可用于氟喹诺酮类药物(FQs)多残留检测方法研究。

Modified EDC method was employed to synthesize norfloxacin（NOR） artificial antigen and New Zealand rabbits were immunized for anti-NOR polyclonal antibody（pAb） production.An indirect competitive ELISA standard curve was established.The developed assay was sensitive and had a linear range from 0.07 to 31.8 ng/mL（R2 = 0.9627）,with IC50 and LOD of respectively 1.5 ng/mL and 0.04 ng/mL.The optimal pH and phosphate ion concentration in assay buffer were determined to be 7.4 mmol/L and 10 mmol/L,respectively,and the concentration adjustments for methanol and NaOH（30 mmol/L） in dilute solutions did not exceed 30% and 10%,respectively.The produced pAb had broad-spectrum specificity and exhibited a high cross-reactivity with ciprofloxacin（83.3%）,pefloxacin（78.9%）,enoxacin（68.2%）,enrofloxacin（51.7%） and lomefloxacin（41.7%）.The results obtained suggest that the established icELISA method be used for the multi-determination of fluoroquinolone residues.