Microsoft Word 赵凯-二校 出4154

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1 6 10 Vol. 6 No Journal of Food Safety and Quality Oct., 2015 赵 1, 凯 2, 贾彦博 3*, 王啸 2, 丁枫芸 2, 肖海龙 3, 黄建萍 3 (1., ; 2., ; 3., ) 摘要 : 目的 ELISA 方法, Balb/c 4, ELISA,,, ELISA 结果, 1:10 7, 1: , ELISA 10 ng/ml, 结论 ELISA, 关键词 : ; ; ; Study on monoclonal antibody and polyclonal antibody preparation and establishment of ELISA detection method of wheat globulin ZHAO Kai 1, 2, JIA Yan-Bo 3*, WANG Xiao 2, DING Feng-Yun 2, XIAO Hai-Long 3, HUANG Jian-Ping 3 (1. School of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou , China; 2. Hangzhou Institute of Calibration and Testing for Quality and Technical Supervision, Hangzhou , China; 3. Hangzhou Institute for Food and Drug Control, Hangzhou , China) ABSTRACT: 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: 10 7, and the polyclonal antibody titer was about 1: 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 基金项目 : ( ) Fund: Supported by Quality and Technical Supervision System Research Major Projects of Zhejiang Province ( ) * 通讯作者 :,, @qq.com *Corresponding author: JIA Yan-Bo, Senior Engineer, Hangzhou Institute for Food and Drug Control, Hangzhou , China @qq.com

2 10, : 4155 ingredients adulteration and allergens in dairy products. KEY WORDS: wheat globulin; monoclonal antibodies; polyclonal antibody; enzyme linked immunosorbent assay 1 引言 [1],, 2%~3% 5%~8% [2], 8 [3],, [4], [5] 2010, GB/T [6],,,,,,,, (PCR) [7] [8] [9] (SDS-PAGE) [10,11] (ELISA),,,, ELISA, 2 材料和方法 2.1 主要试剂和仪器 1500 (BSA) HAT Sigma ; ; RPMI-1640 GIBCO ; (HRP) IgG ; 8 wk Balb/c ; sp2/0 ; (MK3) Thermo, Eppendorf 2.2 试验方法 小麦球蛋白提取与纯化,,, 3% NaCl,,,, 3,, 动物免疫 1:1, 1:1, 4 wk 2, 3 wk 3, 3 wk 4 50 μg/ ELISA, 1:10 5, B 单克隆抗体的纯化与效价测定, 5:1~10:1 sp2/0,, HAT /, 3~4,, , 8~10 d protein G, SDS-PAGE ELISA 多克隆抗体制备与纯化 1:1,,,

3 :1, 4 2, 3 3, 3 4,,, Protein A, ELISA 双抗夹心 ELISA 方法及其灵敏度和特异性,, HRP, ELISA 1 μg/ml, 2, ELISA, P/N 2 3 实验结果 3.2 单克隆抗体 Balb/c ELISA 3,, protein G,, SDS-PAGE 1 1:100 10, 405 nm,,, 2 2, 1: 小麦球蛋白提取与纯化 3%,, SDS-PAGE ( 1), 6, kda, 95% 3% NaCl 2 mg/l,, Fig. 2 2 Titers of wheat globulin monoclonal antibody 1 Fig. 1 Electropherogram of purified wheat globulin and its monoclonal antibody M: Marker; 1: ; 2: M: protein Marker; 1: wheat globulin monoclonal antibody ; 2: wheat globulin 3.3 多克隆抗体,, 3,, Protein A,, 3,, ELISA, 1 1, 1: ELISA 方法的灵敏度,, ELISA 0.5 g/ml,

4 10, : , ELISA, ELISA 3 3, ELISA 10 ng/ml, 表 1 小麦球蛋白多克隆抗体效价 (n=6) Table 1 Titers of wheat globulin polyclonal antibodies (n=6) OD ± ± ± ± ± ± ±0.038 Control 0.103± ELISA Fig. 3 ELISA sensitivities of wheat globulin 3.5 ELISA 方法的特异性 - ( -casein) (barley globulin) (wheat albumin) (wheat gliadin) (zein) (arachin), ELISA,, ELISA,, 实际样品测试 ELISA,, ELISA, ELISA,, 2 4 讨论,,,,, [12], ELISA,,,,, 10 ng/ml, ;, ELISA,,,, 表 2 Table 2 小麦球蛋白 ELISA 方法的特异性和实际样品测试 ELISA specificities of wheat globulin and samples test ELISA : 1- - ; 2- ; 3- ; 4- ; 5- ; 6- ; 7- ; 8- ; 9- ; 10- ; 11-

5 4158 6,,,,,,, ELISA, 参考文献 [1] Taylor SL, Hefle SL. Food allergen labeling in the USA and Europe [J]. Curr Opin Allergy, 2006, 6(3): [2] Moreno FJ. Gastrointestinal digestion of food allergens: Effect on their allergenicity [J]. Biomed Pharm, 2007, 61(1): [3],,. [J]., 2007, 28(8): Mao WX, Gao JY, Chen HB. Research progress of wheat food allergy [J]. Food Sci, 2007, 28(8): [4] Battais F, Pineau F, Popineau Y, et al. Food allergy to wheat: identification of immunoglobulin E and immunoglobulin G-binding proteins with sequential extracts and purified proteins from wheat flour [J]. Clin Exp Allergy, 2003, 33(7): [5] Johansson SG, Hourihane JO, Bousquet J, et al. A revised nomenclature for allergy. An EAACI position statement from the EAACI nomenclature task force [J]. Allergy, 2001, 56(9): [6] GB/T [S]. GB/T Allergenic ingredients in prepackaged foods [S]. [7],,,. PCR [J]., 2011, 50(5): Dong W, Cao JJ, Zheng QZ, et al. Detecting sensitinogen wheat in food by real-time PCR [J]. Hubei Agric Sci, 2011, 50(5): [8],,,. [J]., 2008, 33(12): Li HL, Jiao QN, Huang JR, et al. Precipitation determination of casein and its application for economic adulteration identification in milk [J] Food Sci Technol, 2008, 33(12): [9],,,. [J]., 2005, 31(1): Zhang DS, Pang GC, Gao FG, et al. Application of capillary electrophoresis in determination of casein quantity and detection of adulteration in milk [J] Food Ferment Ind, 2005, 31(1): [10],,,. RP-HPLC [J]., 2009, 30(24): Wang H, Zhang ZG, Chang YZ, et al. Isolation and quantification of main milk proteins in dairy products with reverse-phase HPLC [J]. Food Sci, 2009, 30(24): [11],,,. [J]., 2009, 37(11): Wang J, Zhang QH, Wang ZH, et al. Determination of major bovine milk proteins by reversed phase high performance liquid chromatography [J]. Chin J Anal Chem, 2009, 37(11): [12],,,. β- β- ELISA [J]. ( ), 2013, 39(2): Xiao HL, Zhao K, Lin SJ, et al. Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin [J]. J Zhejiang Univ (Agric & Life Sci Ed), 2013, 39(2): 作者简介 ( 责任编辑 : 李振飞 ) 赵凯, 本科, 主要研究方向为食品安全检测研究 zhaok@hzzjy.net 贾彦博, 高级工程师, 主要研究方向为食品安全检测技术 @qq.com