油菜薹中醚菊酯酶联免疫(ELISA)快速检测方法建立

Rapid determination of ethofenprox in rapeseed stalk by ELISA

为快速有效监控蔬菜（油菜薹）醚菊酯农药残留,采用醚菊酯与牛血清白蛋白偶联物（ethofenproxBSA）抗原免疫新西兰大白兔获得抗醚菊酯多克隆抗体。以醚菊酯与卵清白蛋白偶联物（ethofenprox-OVA）为检测抗原,利用此抗体,进行间接竞争ELISA法试验。通过棋盘格实验确定了最佳抗原包被反应质量浓度为0.25μg/m L,最佳抗原包被条件为37℃孵育2h;一抗最佳稀释度为1∶32 000;最佳p H值与离子浓度分别为6.0和0.16mol/L;最佳封闭试剂与有机溶剂分别为脱脂奶粉和10%甲醇/PBS。在此优化条件下,建立了醚菊酯竞争抑制曲线,抗体的灵敏度（IC50）为0.28μg/m L,检测线性范围（IC20~IC80）为0.079 8~3.578μg/m L。采用该方法对市场抽样进行加标回收测定,平均回收率在75.26%~97.56%之间,实际样品检测间接ELISA与GC/MS检测结果相关性系数达到0.994,灵敏度、精密度和准确度均符合快速检测要求。该法为快速、简便、低耗的醚菊酯免疫检测方法提供了可能。

For rapid and accurate ELISA detection of ethofenprox residue in rapeseed stalks（ vegetable）, polyclonal antibody was obtained from rabbits with ethofenprox - bovine serum albumin（BSA）conjugate（ethofen-prox - BSA）. Based on the antibody,an indirect competitive ELISA was developed with ethofenprox - OVA as the envelope antigen. Assays were performed in the ethofenprox - BSA（0. 25μg / mL）coated ELISA format in which the antibody was 1： 32 000 diluted. The optimized physicochemical factors（pH,ionic strength and blocking solu-tion）in the performance were obtained：blocking reagent was skim milk powder,pH6. 0,and ionic strength was 0. 16mol/ L. Calibration curve was established under the optimal conditions,from which the sensitivity（IC50 ）was 0. 28μg / mL of antibody and the linear working range were IC20 - IC80 of 0. 079 8 - 3. 578μg / mL. Ethofenprox pes-ticides residue in rapeseed stalks collected from markets were screened by the developed ELISA with average recov-ery of 75. 26% - 97. 56% . The correlation coefficient of results obtained by both indirect competitive ELISA and GC / MS method was 0. 994. It concluded a practical ELISA method,and the validation of ethofenprox detection by ELISA and GC - MS in rapeseed stalks was proven.