抗麦草畏多克隆抗体的制备及其间接竞争ELISA检测方法的建立

Preparation of polyclonal antibody against dicamba and establishment of indirect competitive ELISA for dicamba detection

为建立麦草畏的免疫分析方法,采用碳化二亚胺法将麦草畏(DIC)与卵清蛋白(OVA)和牛血清蛋白(BSA)偶联,分别合成免疫原与包被原;用合成的免疫原免疫新西兰大白兔获得抗麦草畏多克隆抗体,该抗体效价为1∶1.28×105,且该抗体与麦草畏的结构类似物2,3,5-三氯苯甲酸、2,3,6-三氯苯甲酸、2-氨基-3,5-二氯苯甲酸的交叉反应率均小于0.1%。以包被原1∶8 000和多抗溶液1∶80 000的稀释倍数为工作浓度,建立麦草畏间接竞争ELISA(ciELISA)检测方法,此方法检测麦草畏的线性范围为1～100ng.mL-1,线性回归方程为y=8.684 4ln x+15.001,R2=0.994 4,最低检测限为IC20=1.779ng.mL-1;玉米粉和面粉空白样品的麦草畏添加回收实验表明,麦草畏添加值为5～30μg.kg-1时,玉米粉中的麦草畏回收率为53.08%～92.37%,面粉的回收率则分别为66.5%～94.63%。

In order to establish the immunoassay method of dicamba（DIC）, the diimine carbonization methods were used to synthesize immunogen and coating antigen by dicamba being conjugated to ovalbumin （OVA） and bovine serum albumin （BSA）, respectively. The polyclonal antibodies against dicamba were prepared through immunizing New Zealand white rabbits with the immunogen. The enzyme-linked immunosorbent assay （ELISA） titer of the antibody was 1 ： 1.28 × 10^5. The percentages of the antibody cross reactivity with other analogues of dicamba （2,3, 5-trichlorobenzoic acid, 2, 3, 6- trichlorobenzoic acid and 2-amino-3, 5-dichlorobenzoic acid） were all less than 0.1%. An indirect competitive ELISA （ciELISA） was developed for dicamba detection, with coating antigen and polyclonal antibody diluted at 1 ： 8 000 and 1 ： 80 000. The lineal range of the assay for detecting dicamba was 1 to 100 ng. mL^-1 , with the detection limit （IC20） of 1. 779 ng. mL^-1 . The regression equation was y = 8. 6844 lnx＋15.001, R^2=0.994 4. When dicamba addition value was from 5 to 30 bag.kg^-1 in the recovery tests, the recovery rate of dicamba in corn meal and wheat flour blank samples ranged from 53. 08% to 92.37%, and 66.5% to 94.63%, respectively.