直接竞争酶联免疫吸附-高效液相色谱法测定烯效唑

摘要

With the high specific affinitive antibody to uniconazole prepared in our lab, the method of antibody-immobilized direct competitive ELISA was employed to determine uniconazole residue in apple. Under the optimized conditions the antibody of 4 mg/L dissolved in 0. 02 mol/L pH7. 6 PB was immobilized on microplate, the peroxidase-hapten conjugate was 105 times diluted in the reaction medium of 0. 02 mol/L pH 6. 8 PBS containing 0. 15 mol/L NaCl, the liner concentrations ranged from 1 mg/L to 10-4 mg/L, the half-maximal inhibition (IC50) was 1. 31 μg/L with relative standard deviation (RSD) of 8. 4%, the IC20 was 0.022 μg/L. The recoveries were 84. 6%-101%, 99. 6% -117% and 80. 8%-92. 8% with the RSD of 8. 6%, 6.9% and 6.7% at the spiked level of 1.0, 0. 1, 0. 01 mg/kg in apple respectively. The limit of detection for ELISA was 0. 024 μg/kg, and the limit of detection for HPLC-ultraviolet visible detector was 0. 25 mg/kg.%应用本实验室制备的对烯效唑具特异性亲和力的多克隆抗体,采用包被抗体直接竞争模式建立烯效唑的酶联免疫吸附分析法.在优化条件下,对烯效唑标样检测的线性范围为1～10-4 mg/L,线性中浓度为1.31 μg/L,5次重复测定的相对标准偏差(RSD)为8.4％；检出限为0.022 μg/L.在苹果中分别添加烯效唑标样1.0,0.1和0.01 mg/kg;直接竞争ELISA法测定的回收率分别为84.6％～101.0％,99.6％～117.0％和80.8％～92.8％；RSD(n=5)分别为8.6％,6.9％和6.7％.ELISA法对苹果中烯效唑的检出限为0.024μg/kg.在同样前处理条件下,高效液相色谱-紫外检测(HPLC-UVD)法对苹果中烯效唑的检出限为0.25 mg/kg,苹果中添加1.00 mg/kg烯效唑,HPLC-UVD法测定的回收率为92.6％.