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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2013, Vol. 39 Issue (2): 222-226    DOI: 10.3785/j.issn.1008-9209.2012.11.617
论文     
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
肖海龙*， 赵凯， 林赛君， 王红青， 潘建红
杭州市质量技术监督检测院，杭州 310019
Preparation of doubleantibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin
XIAO Hailong*, ZHAO Kai, LIN Saijun, WANG Hongqing, PAN Jianhong
Hangzhou Institute of Calibration and Testing for Quality and Technical Supervision, Hangzhou 310019, China
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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15 ng/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.
Abstract: 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∶1×105, and the mice spleen cells and myeloma cells SP2/0 were fused as the routine cellfusion technology. 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 polyclonal antibodies against β-casein and β-conglycinin were also prepared from rabbit serum immunized by antigens, respectively. The double antibody sandwich ELISA for β-casein and β-conglycinin were successfully established by optimized parameters. The results showed that the titers of purified monoclonal antibodies of β-casein and β-conglycinin were over 1∶1×107, and the polyclonal antibody titers of both were about 1∶2×105. 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 methods established for β-casein and β-conglycinin are sensitive and specific, and can afford a theoretical foundation for developing rapid, accurate and lowcost screening methods for the detection of adulteration and allergens.
出版日期: 2013-03-20
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肖海龙*
赵凯
林赛君
王红青
潘建红

引用本文:

肖海龙*， 赵凯， 林赛君， 王红青， 潘建红. 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[J]. 浙江大学学报(农业与生命科学版), 2013, 39(2): 222-226.

XIAO Hailong*, ZHAO Kai, LIN Saijun, WANG Hongqing, PAN Jianhong. Preparation of doubleantibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(2): 222-226.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.11.617    或    http://www.zjujournals.com/agr/CN/Y2013/V39/I2/222

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