大豆球蛋白间接竞争ELISA检测方法的建立

CONSTRUCTION OF INDIRECT COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR SOYBEAN GLYCININ DETECTION

建立了大豆球蛋白间接竞争酶联免疫检测法（ELISA）,为食品中主要的大豆蛋白过敏原的检测提供技术基础.以纯品大豆球蛋白免疫新西兰大白兔,自制得到兔抗大豆球蛋白的多克隆抗体,并以大豆球蛋白包被抗原、自制抗体作为一抗、辣根过氧化物酶标记的羊抗兔IgG为酶标二抗,四甲基联苯胺（TMB）显色液为底物,建立了大豆过敏原中大豆球蛋白的间接竞争ELISA检测方法.确定了抗原的包被浓度为0.025μg/mL,一抗最佳稀释度为1∶3 200,酶标二抗稀释度为1∶10 000.检测结果达到板内误差3.82%,板间误差10.22%,在允许的误差范围之内;其检测的线性范围为0.01~0.05μg/mL.结果表明本试验所建的大豆球蛋白间接ELISA方法具有一定的重复性和灵敏度,可用于大豆蛋白制品过敏原的检测.

An indirect competitive enzyme-linked immunosorbent assay（ELISA） was constructed to detect the main allergen glycinin in foods. A polyclonal antibody of rabbit anti-glycinin was prepared by using glycinin to immunize New Zealand rabbits. The indirect competitive ELISA method was constructed by coating an antigen with glycinin,using the polyclonal antibody as the primary antibody,using HRP（horse radish peroxidase）-marked goat anti rabbit IgG as IgG-HRP,and using tetramethyl benzidine（TMB）substrate. Results showed that the optimum conditions were as follows： the antigen coating concentration 0.025 μg/mL,the primary antibody dilution degree 1 ∶3200,and the IgG-HRP dilution degree 1： 10000; the in-plate error and the inter-plate error were 3.82% and 10.22%,which were in the allowable error range; and the detection linear range was 0.01 to0.05 μg/mL. The indirect competitive ELISA method constructed in the paper has good reproducibility and sensitivity,and can be used for allergen detection of soy protein products.