生物素-亲和素放大酶联免疫吸附法测定呋喃 西林代谢物

Determination of nitrofurazone metabolite by biotin-avidin enzyme-linked immunosorbent assay

中文关键词:  兽药  呋喃西林  氨基脲  生物素-亲和素放大酶联免疫吸附法

英文关键词:veterinary medicine  nitrofurazone  semicarbazide  biotin-avidin enzyme linked immunosorbent assay

中文摘要:

      目的 建立竞争生物素-亲和素放大酶联免疫吸附测定法(biotin-avidin enzyme linked immumosobent assay, BA-ELISA)检测呋喃西林代谢物。方法 采用棋盘法确定单克隆抗体和包被原的最佳稀释倍数, 通过单因素试验优化辣根过氧化物酶标记的链霉亲和素(streptavidin-horseradish peroxidase, SA-HRP)稀释倍数、封闭液浓度、竞争反应时间、生物素标记羊抗鼠二抗(biotinylated goat anti-mouse ImmunoglobulinG, Biotin-IgG)孵育时间、SA-HRP孵育时间和显色时间, 建立标准曲线, 并对方法的灵敏度、特异性、准确度和精密度进行方法学评价。结果 最佳实验条件为: 包被原稀释倍数为1:800, 单克隆抗体稀释倍数为1:12800, SA-HRP稀释倍数为1:8000, 封闭液为1%的脱脂乳, 竞争反应时间为30 min, Biotin-IgG孵育时间为30 min, SA-HRP孵育时间为60 min, 显色时间为15 min; 在优化条件下, 线性方程为Y=-0.3299X+0.4272 (r2=0.990), IC50为0.601 ng/mL, 线性范围为0.074~4.883 ng/mL; 加标回收率为88.8%~98.5%, 批内变异系数(coefficient of variation, CV)和批间CV分别为1.2%~3.6%和2.5%~5.1%, 建立的方法与其他结构类似物及衍生化试剂的交叉反应率(cross reactivity, CR)均小于0.1%。BA-ELISA与高效液相色谱-串联质谱法的回收率均在85%~115%范围内。

英文摘要:

      Objective To establish a method for determination of nitrofurazone metabolite by biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA). Methods The optimal dilutions of monoclonal antibody and coating antigen were determined by chequerboard titration. The effects of the dilution ratio of streptavidin-horseradish peroxidase (SA-HRP), the concentration of sealing fluid, the competitive reaction time, the incubation time of biotinylated goat anti-mouse IgG(Biotin-IgG), SA-HRP incubation time and chromogenic reaction time were optimized by single-factor experiments. The standard curve was set up. The degree of sensitivity, specificity, precision and accuracy about the method were evaluated. Results The result of optimized reaction conditions were as follows: fold dilution of coating antigen was 1:800, fold dilution of monoclonal antibody was 1:12800, fold dilution of SA-HRP was 1:8000, sealing fluid was 1% dried skim milk, competitive time was 30 min, Biotin-IgG reaction time was 30 min, SA-HRP reaction time was 60 min and chromogenic reaction time was 15 min. The linear detection range was Y=-0.3299X+0.4272 (r2=0.990), IC50 was 0.601 ng/mL, the linear range of the presented method was 0.074~4.883 ng/mL, the recoveries in negative samples were 88.8%~98.5% and the intra-assay and inter-assay coefficients of variation were 1.2%~3.6% and 2.5%~5.1%, respectively. The cross-reactivity values were lower than 0.1% with other structural analogues and derivatives. The recoveries of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and BA-ELISA were all in the range of 85%~115%. Conclusion This method has high sensitivity, high specificity and good reproducibility, which can be used for rapidly screening nitrofurazone metabolites in animal tissues.