小麦球蛋白单克隆和多克隆抗体制备及建立酶联免疫吸附法快速检测技术

目的建立小麦球蛋白的ELISA检测技术,用于蛋白质掺假以及过敏原成分的快速检测。方法从麦胚粉中提取球蛋白,抗原免疫Balb/c小鼠进行4次免疫后,通过脾脏细胞杂交瘤技术及间接ELISA筛选制备单克隆抗体,同时制备兔抗小麦球蛋白的多克隆抗体。通过棋盘滴定法,初步确定单克隆抗体和多克隆抗体的最佳工作浓度,建立双抗夹心ELISA。结果通过免疫和杂交瘤技术获得了抗小麦球蛋白的单克隆抗体,纯化后抗体的效价均达到1:107,通过免疫兔制备的多克隆抗体经纯化后效价在1:2.4×105左右,所建立的双抗体夹心ELISA方法最低检测限为10 ng/m L,与其他物种的蛋白不发生交叉反应。结论本文建立的双抗体夹心ELISA方法具有良好的特异性和灵敏度,为建立乳品中小麦成分掺假及过敏原成分快速检测提供理论依据。

Study on monoclonal antibody and polyclonal antibody preparation and establishment of ELISA detection method of wheat globulin

Objective To establish an immunoassay method of wheat globulin for the rapid detection of allergen and potential protein adulteration. Methods Globulin was extracted and purified from wheat germ powder. Balb/c mice were immunized for 4 times, and the mice spleen cells and myeloma cells SP2/0 were fused as the routine cell-fushion technology. The monoclonal antibodies were obtained by immune and hybridoma technology after purification of ascites. The polyclonal antibody against wheat globulin was also prepared from rabbit serum immunized by antigen. The double antibody sandwich ELISA for wheat globulin was successfully established by optimizing parameters. Results The results showed that the titer of purified monoclonal antibody of wheat globulin was over 1: 107, and the polyclonal antibody titer was about 1:2.4×105. The minimum detection limits of enzyme linked immunosorbent assay (ELISA) kit was about 10 ng/mL, and no cross reaction was observed among proteins of different species. Conclusion The double antibody sandwich ELISA method was sensitive, specific, and provided a theoretical foundation for detection of wheat ingredients adulteration and allergens in dairy products.