牛奶β-酪蛋白和大豆β-伴球蛋白双抗制备及夹心ELISA快速定性检测技术的建立  

Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin

作　　者：肖海龙[1] 赵凯[1] 林赛君[1] 王红青[1] 潘建红[1] 

机构地区：[1]杭州市质量技术监督检测院,杭州310019

出　　处：《浙江大学学报（农业与生命科学版）》2013年第2期222-226,共5页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：浙江省质量技术监督系统科研计划重大资助项目(20100107)

摘　　要：采用牛奶β-酪蛋白和大豆β-伴球蛋白分别免疫BALB/c小鼠,4次免疫后,通过脾脏细胞杂交瘤技术及间接酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)筛选制备单克隆抗体,同时分别制备兔抗β-酪蛋白和β-伴球蛋白多克隆抗体;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用于乳品掺假以及牛奶、大豆过敏原成分的快速定性检测.结果表明:通过免疫和杂交瘤技术分别获得了抗β-酪蛋白和β-伴球蛋白的单克隆抗体,纯化后2种抗体的效价均达到1∶1×107,通过免疫兔制备的2种多克隆抗体经纯化后效价在1∶2×105左右;所建立的双抗夹心ELISA方法的最低检测限为15ng/mL,与其他物种的蛋白不发生交叉反应,具有良好的特异性.这为建立乳品掺假及过敏原成分快速检测奠定了基础.Protein adulteration and allergens are the two major food safety issues, and can pose health threats to consumers. One of the effective precautions is extensive test, which needs simple, rapid and low-cost test method. Present methods including sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, high performance liquid chromatography （HPLC） and liquid chromatography-mass spectrometry （LC-MS）/MS are not acceptable for consumers due to expensive instruments. Immunological technique is a rapid method for screening and is suitable for consumers to use. β-casein and β-conglycinin are not only the major proteins of milk and soybean, but also important food borne allergens. In this study, the monoclonal antibodies and polyclonal antibodies against β-casein and β-conglycinin were prepared and the enzyme-linked immunosorbent assay （ELISA） kits were established for detecting adulteration and allergens. BALB/c mice were immunized four times with purified antigens adding adjuvant or not until the serum titer achieved 1 ： 1 × 10^5 , and the mice spleen cells and myeloma ceils SP2/0 were fused as the routine ceil-fusion teehnol＇ogy, Positive cells were screened for 3-4 times with indirect ELISA by coating purified antigens, and were injected into mice peritoneal. The monoclonal antibodies were obtained after the purification of ascites. The polyelonal antibodies against β-casein and β-conglyeinin were also prepared from rabbit serum immunized by antigens, respectively. The double antibody sandwich ELISA for β-casein and β-eonglyeinin were successfully established by optimized parameters. The results showed that the titers of purified monoclonal antibodies of β-casein and β-eonglyeinin were over 1 ： 1× 10^7 , and the polyelonal antibody titers of both were about 1 ： 2 × 10^5 . The minimum detection limits of both ELISA kits were about 15 ng/mL, and no cross reaction were observed among the proteins of different species. In conclusion, the double antibody sandwich ELISA m

关 键 词：Β-酪蛋白 β-伴球蛋白 单克隆抗体 酶联免疫吸附测定 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]