沙拉沙星、二氟沙星直接竞争化学发光酶联免疫检测方法的建立

Establishment of a sarafloxacin and difloxacin direct competition chemiluminescence enzyme-linked immunosorbent assay method

为快速检测食源性动物中沙拉沙星及二氟沙星的药物残留,将沙拉沙星单克隆抗体(SAR-Ab)与辣根过氧化物酶(HRP)偶联得到酶标抗体(McAb-HRP),从而建立直接竞争化学发光酶联免疫(dc-CLEIA)检测方法。结果显示:经紫外扫描鉴定,McAb-HRP偶联成功;以包被抗原(SAR-1-OVA)浓度1.25μg/mL,McAb-HRP稀释比1∶8000作为最佳工作条件,从而建立了dc-CLEIA分析法;精密度的平均批内批间变异系数均低于7%;建立的标准曲线呈线性关系,线性范围为0.312~20 ng/mL,回归方程式y=-0.373x+0.688(R^(2)=0.985),经计算其灵敏度IC_(50)为3.19 ng/mL;除二氟沙星外与其他氟喹诺酮类药物的交叉反应率均低于8%,与其他种类的抗生素均无交叉反应;沙拉沙星和二氟沙星在鸡肉样品中的最低检出限分别为1.25、1.36μg/kg;沙拉沙星和二氟沙星在鸡肉样品中的添加回收率分别为88.2%~108.3%、93.36%~102.7%,且其变异系数均≤13.4%。结果表明:此方法不仅快速且灵敏度高,可作为沙拉沙星及二氟沙星药物残留的定量检测方法。

For rapid detection of sarafloxacin and difloxacin drug residues in food-borne animals,a sarafloxacin monoclonal antibody was coupled with horseradish peroxidase(HRP)to obtain an enzyme-labeled antibody(McAb-HRP),for the purpose of establishing a direct competition chemiluminescence enzyme-linked immunoassay(dc-CLEIA).The results showed that the McAb-HRP coupling was successfully identified by ultraviolet scanning.The best conditions were the coating antigen(SAR-1-OVA)concentration of 1.25μg/mL and the McAb-HRP dilution ratio of 1∶8000 for establishing a dc-CLEIA analysis method.The average intra-assay and inter-assay coefficient of variation of precision was lower than 7%.The established standard curve showed a linear relationship,the linear range was 0.312-20 ng/mL,the regression equation was y=-0.373x+0.688(R^(2)=0.985),and the sensitivity IC_(50) was calculated to be 3.19 ng/mL.The cross-reaction rate with other fluoroquinolone drugs except Difloxacin was lower than 8%,and there was no cross-reaction with other kinds of antibiotics.The lowest detection limits of sarafloxacin and difloxacin in chicken samples were 1.25 and 1.36μg/kg,respectively.The recovery rates of sarafloxacin and difloxacin in chicken samples were 88.2%-108.3%,and 93.36%-102.7%,respectively.Their coefficient of variation both were≤13.4%.The above results indicated that the present method was not only fast and sensitive,but could also be used as a quantitative method for detecting sarafloxacin and difloxacin drug residues.