呋喃它酮代谢物间接竞争酶联免疫检测方法的建立

Establishment of an indirect competitive enzyme linked immunosorbent assay detection method for furaltadone metabolite

中文关键词:  兽药残留  呋喃它酮  酶联免疫检测法

英文关键词:veterinary medicine residue  furaltadone  enzyme linked immunosorbent assay

中文摘要:

      目的 建立呋喃它酮代谢物5-甲基吗啉-3-氨基-2-唑烷基酮(5-morpholine-methyl-3-amino- 2-oxazolidinone, AMOZ)间接竞争酶联免疫检测法。方法 通过衍生化反应制备了人工抗原CPAMOZ并用活化酯法将其与鸡卵清蛋白(ovalbumin, OVA)连接成为包被原CPAMOZ-OVA。对包被原和单克隆抗体的最佳工作浓度、封闭液浓度、竞争时间、辣根过氧化物酶标记羊抗鼠二抗(horseradish peroxidase labelled goat antimouse IgG conjugate, HRP-IgG)、孵育时间和显色反应时间进行优化, 并对方法灵敏度、特异性、精密度和准确度进行方法学评价。结果 最优条件下, 包被原和单克隆抗体稀释倍数分别为500倍和2000倍, 封闭液浓度为2%, 竞争时间为30 min, 二抗孵育时间为30 min, 显色时间为15 min。所建立标准曲线的对应线性方程是Y= ?0.439X+0.670(r2=0.992), IC50为2.439 ng/mL, 线性范围为0.506~11.765 ng/mL, 回收率为86.7%~90.1%, 批内和批间变异系数分别为1.4%~5.8%和2.1%~4.9%。结论 该方法特异性强, 准确性、精密度和重现性良好, 可用于对AMOZ的快速筛查。

英文摘要:

      Objective To establish a method for the determination of furaltadone metabolite (5-morpholine- methyl-3-amino-2-oxazolidinone, AMOZ) by indirect competitive enzyme linked immunosorbent assay. Methods The artificial antigen CPAMOZ was prepared by the derivation reaction and the activation ester method was used to connect it with ovalbumin (OVA) to the original CPAOZ-OVA. The working concentration of coating antigen and monoclonal antibodies, blocking concentration, competition time, incubation time of HRP-IgG and colouration time were optimized, besides, the sensitivity, specificity, precision and accuracy of this method were evaluated. Results In the optimal conditions, the coating antigen and monoclonal antibody dilution were 500 times and 2000 times, respectively, the concentration of blocking liquid was 2%, competition time was 30 min, colouration time was 15 min, and incubation time was 30 min. The corresponding linear equation of the standard curve was Y= ?0.439X+0.670 (r2=0.992), and the IC50was 2.439 ng/mL, the linear range was 0.506~11.765 ng/mL, the recovery rate was 86.7%~90.1%, the intra-and inter-assay coefficients of variation were 1.4%~5.8% and 2.1%~4.9%, respectively. Conclusion This method is specific, accurate, accurate and reproducible, which can be used for rapid screening of AMOZ.