College of Veterinary Medicine, Northwest A&F University, Yangling, China.
J Zhejiang Univ Sci B. 2012 Jul;13(7):545-54. doi: 10.1631/jzus.B1200001.
Modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesize the artificial antigen of norfloxacin (NOR), and New Zealand rabbits were used to produce anti-NOR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curve was established. This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml, with the half maximal inhibitory concentration (IC(50)) and limit of detection (LOD) values of 2.7 ng/ml and 0.06 ng/ml, respectively. The produced pAb exhibited high cross-reactivity to fluoroquinolones (FQs) tested, and the IC(50) values to enoxacin, ciprofloxacin, and pefloxacin were 3.1, 3.4, and 4.1 ng/ml, respectively. It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10% and 30%. When spiked in milk at 5, 20, and 50 ng/ml, the recoveries for NOR, enoxacin, ciprofloxacin, and pefloxacin ranged 90.5%-98.0%, 84.0%-95.2%, 94.0%-106.0%, and 89.5%-100.0%, respectively. The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.
改良的 1-乙基-3-(3-二甲基氨基丙基)碳化二亚胺（EDC）法被用于合成诺氟沙星（NOR）的人工抗原，并使用新西兰兔生产抗 NOR 多克隆抗体（pAb）。基于棋盘滴定法，建立了间接竞争酶联免疫吸附测定（icELISA）标准曲线。该测定法具有较高的灵敏度，工作范围为 0.12 至 68.40ng/ml，半数抑制浓度（IC（50））和检测限（LOD）值分别为 2.7ng/ml 和 0.06ng/ml。所产生的 pAb 对测试的氟喹诺酮类（FQs）具有较高的交叉反应性，对恩诺沙星、环丙沙星和培氟沙星的 IC（50）值分别为 3.1、3.4 和 4.1ng/ml。此外，测定缓冲液中的 NaOH 和甲醇浓度不应高于 10%和 30%。当在牛奶中以 5、20 和 50ng/ml 的浓度添加时，NOR、恩诺沙星、环丙沙星和培氟沙星的回收率分别为 90.5%-98.0%、84.0%-95.2%、94.0%-106.0%和 89.5%-100.0%。结果表明，该基于特异性 pAb 的 icELISA 可用于动物源性产品中 FQ 残留的初步筛选。
