间接竞争酶联免疫吸附分析法测定土壤和面粉中的异丙隆

Development of an Indirect Competitive ELISA for the Determination of Isoproturon in Soil and Flour

采用自制的抗异丙隆多克隆抗体建立了测定异丙隆残留的间接竞争酶联免疫分析方法,优化的包被抗原和抗体的浓度分别为0.1和0.016mg·L-1。结果表明,6种常用的与异丙隆结构相似的脲类除草剂的交叉反应都小于0.6%,标准曲线的线性范围为3.42～121.33ng·mL-1,线性相关系数r2=0.9410,方法的检测限为3.42ng·mL-1。利用建立的间接竞争ELISA方法检测土壤和面粉样品中的异丙隆,测得土壤中异丙隆的加标回收率为100%～108%,变异系数为3%～6%;面粉中异丙隆的加标回收率为81%～84%,变异系数为5%～7%。

Isoproturon, 3-（4-isopropylphenyl）-1,1-dimethylurea, is the most common representative of the urea herbicides for pre- and post-emergence control of black grass, silky bent grass, wild oats, annual meadow grass, ray grass, and many broadleaf weeds in spring and winter wheat. The urea herbicides are slightly water-soluble and can easily migrate through the soil to crops and thus enter the food chain. In this paper, a specific conjugate of 1-（5-carboxypentyl）-3- （4-isopropylphenyl）-1-methylurea and bovine serum albumin （BSA） was used as immunogen, with which we successfully obtained a high-titer anti-isoproturon polyclonal antibody （pAb） by immunization of rabbits. A sensitive immunochemical method for the determination of isoproturon based on indirect competitive enzyme-linked immunosorbent assay （ELISA） was established using the pAb. The optimal concentrations of the coating antigen [1-（3-carboxypropyl）-3-（4-isopropylphenyl）-1- methylurea-OVA] and the pAb used in the ELISA were 0.1 mg·mL^-1 and 0.016 mg·mL^-1, respectively. Blocking agents were evaluated to hinder nonspecific sorption of an antibody onto the less-coated plate. Compared with OVA, skim milk as a blocker showed a higher sensitivity for the ELISA. The cross-reactivities of other six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.6%. The linear range of the calibration curve was 3.42-121.33ng·mL^-1 （r^2=0.941 0）. The limit of detection for the indirect ELISA was 3.42 ng·mL^-1. The recoveries for the spiked soil and flour were between 100%-108% and 81%-84%, respectively. The coefficients of variation were 3%- 6% and 5%-7%, respectively.