利用荧光免疫层析技术定量检测相思子毒素

A fluorescent microsphere immunochromatography method for quantitative detection of abrin

目的利用荧光免疫层析技术,研发定量检测相思子毒素的试纸条。方法采用亲和层析法纯化相思子种子,用凝胶过滤层析纯化天然相思子毒素,制备鼠抗相思子毒素单克隆抗体和兔抗相思子毒素多克隆抗体,将特异性单抗结合荧光微球,兔多抗包被检测线,羊抗鼠二抗包被质控线,建立相思子毒素双抗体夹心定量检测方法及其试纸条,并对该试纸条进行性能评价。结果经纯化获得分子量31 k Da的相思子毒素A链和33 kDa的B链。研发的荧光免疫层析试纸条配合荧光免疫检测仪在0~50 ng/ml的检测范围内拟合曲线R^2=0.990,线性范围为1~10 ng/ml,最低检出限为1 ng/ml。批间、批內重复性均小于15%,常温稳定性14个月,采用动物血清和食物模拟样本检测特异性良好。结论开发的荧光免疫层析试纸条可以用于食品和环境中相思子毒素的检测。

Objective To develop an immunochromatographic reagent for the detection of abrin. Methods The native abrin was purified by affinity chromatography,and then SephacrylTMS-200 chromatography was carried out to remove lectins according to the molecular weight difference between abrin and lectin. The purified native abrin pro-tein was used to immunize Kunming mice and rabbit to obtain the monoclonal antibody and polyclonal antibody.Combining the labeling technology,the specific monoclonal antibody were conjuncted with fluorescence nanoparticles,abrin rabbit polyclonal antibodies were sprayed on the test zone,and goat anti-mouse IgG was sprayed on the control zone to develop lateral flow immunochromatographic strips. Results The purified protein showed two bands which molecular weight was 31 kDa and 33 kDa subjected to SDS-PAGE,which were equal as abrin toxin A chain and B chain. In the quantitative test,the fluorescence intensity was measured by a portable strip reader and a standard curve was obtained from 0-50 ng/ml(R^2≥0.990),and the linear rangeand was from 1-10 ng/ml. The sensitivity of immunofluorescence detection stripes was 1 ng/ml. Both the variation coefficients were less than 15%,the stability at room temperature was 14 months. Finally,animal serum and several common foods were used to simulate the environmental samples,the developed method showed no cross-reaction. Conclusion The proposed strip assay could be rapid screening tools for rapid detection of the abrin in food and environmental samples.