诺氟沙星单克隆抗体的制备及初步应用

Preparation and application of monoclonal antibody against norfloxacin

采用优化的碳二亚胺法制备诺氟沙星(NOR)人工抗原,紫外扫描和红外光谱图表明人工抗原偶联成功。采用细胞融合技术筛选抗NOR单克隆抗体(NORmAb)杂交瘤细胞株,体内诱生腹水法制备NORmAb,建立间接竞争ELISA(icELISA)标准曲线。结果表明,筛选的3株杂交瘤细胞分泌的抗体为IgG1亚型,具k轻链;建立的icELISA标准曲线在PBS中的的线性检测范围为0.04～43.6ng/mL,IC50值为0.62ng/mL,检测限(LOD)为0.02ng/mL;抗体与环丙沙星(87.2%)、培氟沙星(76.9%)、依诺沙星(66.8%)、恩诺沙星(48.6%)和洛美沙星(36.6%)有较高的交叉反应率;禽肉基质中NOR添加回收率满足检测要求。

Modified EDC method was employed to synthesize the artificial antigen of norfloxacin（NOR）,which was proved by Ultraviolet absorbance and IR spectra.Cell fusion technology was used to screen anti-NOR hybridoma lines,and monoclonal antibodies（mAbs）were produced by using ascitic fluid in mice.Based on this,an indirect competitive ELISA standard curve was established.This assay was sensitive and had a linear range from 0.04～43.6ng/mL（R2=0.9655）,with IC50 and LOD values of 0.62ng/mL and 0.02ng/mL,respectively.The produced mAbs were of the IgG1 isotype with a k light chain,and mAb from N7B2 exhibited high cross-reactivities to ciprofloxacin（87.2%）,pefloxacin（76.9%）,enoxacin（66.8%）,enrofloxacin（48.6%）and lomefloxacin（36.6%）.The spiking tests indicated that the established icELISA method could be used for determination of NOR residue in poultry.