氯黄隆酶联免疫吸附分析技术研究

Studies on Enzyme-Linked Immunosorbent Assay of Chlorsulfuron

对邻氯苯磺酰胺进行衍生合成半抗原 ,并与载体蛋白质共价偶联制备突出氯黄隆分子特异性部分的合成抗原 ,以合成抗原免疫兔获得对氯黄隆具高亲合力的抗血清。采用硫酸铵盐析和 DEAE纤维素反相吸附法分离纯化抗体 ,用辣根过氧化物酶以改良的过碘酸钠法标记抗体、以混合酸酐法标记半抗原。在此基础上建立了对氯黄隆具高度特异性的间接竞争 ,包被抗体、包被抗原直接竞争酶联免疫吸附分析技术。在优化条件下 ,氯黄隆测定的线性浓度范围为 1 0 0 ～ 1 0 3 ng/ m L,检出限 <0 .1 ng/ m L。

A hapten(CSH)derived from 2 chloro phenylsulfonylamide(CPSA) was conjugated to a carrier protein to prepare synthetic antigens in which the specific part of chlorsulfuron was protruded. The antiserum with high affinity to chlorsulfuron was obtained from New Zealand white rabbits by using synthetic antigen as an immunogen. The polyclonal antibodies were separated from antiserum by salting out method and purified by reversed phase adsorption of DEAE cellulose. The horseradish peroxidase (HRP) tagged antibody was prepared by means of modified NaIO 4 method and the HRP tagged hapten was prepared by means of mixed anhydride method. Based on these, the indirect competitive and antibody or antigen coating direct competitive enzyme linked immunosorbent assays (ELISA), which are highly specific to chlorsulfuron, were established. Under optimized conditions, the linear concentrations ranged from 10 0 to 10 3 ng/mL and the detection limits were lower than 0.1 ng/mL for the determination of chlorsulfuron. Some analogues of chlorsulfuron, such as CPSA, metsulfuron methyl, tribenuron and other common used sulfonylurea herbicides didn't interfere with the analysis of chlorsulfuron.