定量检测转基因植物蛋白Cry1Ac的双抗体夹心ELISA方法建立

Double-antibody Sandwich ELISA for the Quantitative Detection of Cry1Ac Protein in Transgenic Plants

以典型且研究较多的抗虫CrylAc基因表达的CrylAc蛋白为检测目标,制备其单克隆抗体及辣根过氧化物酶标记的单克隆抗体,在优化抗体纯化方案,获得纯化抗体的基础上,以CrylAc单克隆抗体为包被抗体,以辣根过氧化物酶标记的CrylAb单克隆抗体为检测抗体,建立了CrylAc蛋白的双抗体夹心酶联免疫吸附分析(ELISA)检测法,并用于玉米中CrylAc蛋白含量的检测。结果显示,所建立的双抗体夹心ELISA法稳定性较好,测定变异系数在3%以内;在10 ng/mL^200 ng/mL的浓度范围内,线性回归方程为+=0768.0x0114.0y(R2=0.9989),检测限为9.49 ng/mL;对玉米样品提取液中Cry1Ac蛋白含量的测定回收率在102.5%~103%范围内。本研究所建立的双抗体夹心ELISA法为玉米中Cry1Ac蛋白的定量检测提供了有效的手段,可作为一种潜在的检测方法用于转基因产品的检验检疫中,在出入境检验检疫工作中也有较高的应用价值。

In this study, CrylAc protein, encoded by insect-resistance gene CrylAc, was selected as the target antigen and its monoclonal antibody and horseradish peroxidase labeled anti-monoclonal antibody were prepared. Using optimized antibody purification techniques, the purified antibody was obtained and double-antibody sandwich enzyme-linked immunosorbent assay（ELISA） was developed for detection of CrylAc protein in corn. CrylAc monoclonal antibody was used as the coating antibody and horseradish peroxidase-labeled CrylAb monoclonal antibody was used as the detection antibody. The results showed that the newly developed ELISA had good stability and the coefficient of variation was within 3%. In the concentration range of 10~200 ng/mL, the linear regression equation was y = 0.0114 x ＋ 0.0768（R2 = 0.9989）, while the detection limit of the assay was 9.49 ng/mL. The recoveries of Cry1 Ac in corn extract ranged from 102.5% to 103%. The established sandwich ELISA provides an effective method for the quantitative detection of Cry1 Ac protein in corn and is therefore a promising detection method for the inspection and quarantine of transgenic products and may have high application value in entry-exit inspection and quarantine.