水产品中孔雀石绿BA-ELISA检测方法的建立及应用

Establishment and application of BA-ELISA for malachite green detection in aquatic products

建立了基于生物素-链霉亲合素放大的酶联免疫吸附法(BA-ELISA)检测水产品中的孔雀石绿(MG)含量的方法。优化了包被原和生物素化抗体的工作浓度,以及封闭液、温育时间、pH值和盐离子强度等实验条件。结果显示,最佳实验条件为:包被原和生物素化抗体的稀释倍数均为1∶ 2 000,封闭液为1%明胶,抗原抗体反应时间为 60 min ,缓冲液的pH值为7.0,盐离子强度为0.1 mol/L。在此条件下,该方法的线性范围为0.05~20.00 ng/mL,检出限(LOD)为0.037 μg/kg,回收率为83.5%~115.0%,变异系数( CV )为4.53%~13.80%。将该方法用于实际样品的分析检测,检测结果与LC/MS相比,具有较好的相关性。上述结果表明,本实验建立的方法灵敏度高,特异性和稳定性较好,可用于水产品中MG的分析筛查。

A biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) was established for malachite green (MG) detection in this study. The experimental conditions were optimized, including working concentration of coating antigen and biotinylated antibody, blocking buffer, incubation time, pH value, and salt ions intensity. As a result, the optimal conditions were as following: the dilution multiples of coating antigen and biotinylated antibody were 1∶ 2 000, the blocking buffer was 1% gelatin, the reaction time was 60 min, the pH value was 7.0, and the salt ions intensity was 0.1 mol/L. Under the optimal conditions, the linear range of BA-ELISA was 0.05 ng/mL -20.00 ng/mL, the limited of detection (LOD) was 0.037 μg/kg, the recovery was 83.5%-115.0%, and the coefficient of variance ( CV ) was 4.53%- 13.80 %. The method was used for the detection of MG in actual positive samples, and was better correlated with LC/MS. The developed method had high sensitivity, good specificity and stability, and can be used for the analysis and screening for MG in aquatic products.