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1 3 4 Vol. 3 No Journal of Food Safety and Quality Aug., 2012 β- 张海英 1, 刘旸 1, 吴冬雪 1, 梁靖 1, 张霞 1, 2* (1., ; 2., ) 摘要 : 目的 β- ELISA 方法 β-, ELISA 结果 ELISA β-, 结论 ELISA 关键词 : β- ; ; Comparison of two detection methods of β-lactoglobulin allergens in food ZHANG Hai-Ying 1, LIU Yang 1, WU Dong-Xue 1, LIANG Jing 1, ZHANG Xia 1, 2 (1. Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin , China; 2. College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin , China) ABSTRACT: Objective To compare the detection effect of two kinds of β-lactoglobulin ELISA kits on the market. Methods The recovery, precision and specificity of the two brands of ELISA kits were compared by adding positive ingredients, such as skim milk powder and casein. Results ELISA kits could detect casein content in a variety of foods. The recovery, precision and othe,r performance could meet the relevant detection limit requirements. Conclusion The method is easy to operate and does not require any complex equipments. It is applicable to rapid screening for a large number of samples, and a variety of primary laboratory food quality monitoring KEY WORDS: β-lactoglobulin; allergen; ELISA 1 引言, IgE IgE, 0.1%~7.5% [1-2],,,,, [3], -- [4] β-, 10%, 50%, 36 kda, Lipoca1in, 162 β-,,,,, [5] 基金项目 : : ( ) * 通讯作者 :,,, zhangxia_75@126.com

2 296 3, - :, (ELISA); DNA [6-9] ELISA, PCR,,, PCR ELISA, β- ELISA 2 材料与方法 2.1 材料检测试剂盒 A B β- ELISA, ElisaSystems R-biopharm 阳性添加物 () β- (L-8005, Sigma ); β-( IFP ) 食品基质 β- ; ( MPBio ) (sigma ) α- (sigma ); 2.2 方法技术细节比较准确度试验添加试验, β- [10] β- : 10 g 90 g 100 ml, 10% 1 mg/kg, β-: 1 g β- 9 g 10 ml, 10%β- 0.1 mg/kg β-, β ELISA 检测, : (%)=/ 100%, A B β mg/kg,, mg/kg ; β mg/kg 精密度试验 (CV%) β-, mg/kg,, 3, (CV%) CV%: CV(%)= SD/X 100%, SD, X 特异性试验 α- 100 mg/kg 3 结果 3.1 技术细节比较 A B, 1

3 4, : β- 297 Table 1 表 1 试剂盒技术细节比较 Comparison of the technical details of kits A B,, (, ), () min 30 min 30 min 165 min 0.1~1.0 mg/kg 0.01~0.81 mg/kg β- β- 0 mg/kg, 0.10 mg/kg, 0.25 mg/kg, mg/kg, 0.01mg/kg, 0.03 mg/kg, 0.09 mg/kg, mg/kg, 1.00 mg/kg 0.27 mg/kg, 0.81mg/kg β- β-,,, B, 165 min : (1) : A β-, ; B β- (2) : A β- 0.1~1.0 mg/kg; B β- 0.01~0.81 mg/kg,, 50, 5 mg/kg 3.2 准确度试验 2,, 2~ 3 A β mg/kg, 78%~110%, ; B 0.01~0.81 mg/kg, 50, 5 mg/kg, 86.1%~116.6% 3.3 精密度试验 mg/kg β-,, 4 4 A 8%, B 12%,, 3.4 特异性试验, B,, β- A,,, ( ) β-, β- 4 讨论 ELISA,, ELISA ELISA,,,,, FDA 2004 (FALCPA),

4 298 3 mg/kg 表 2 脱脂奶粉在三种基质中回收率范围 (n=18) Table 2 The recovery rate range of skimmed milk powder in the three matrices(n=18) β- A B (mg/kg ) β- mg/kg (%) β- *mg/kg (%) ~ ~ / ~ ~ / ~ ~ / ~ ~ * : B mg/kg 表 3 β- 乳球蛋白在三种基质中回收率范围 (n=18) Table 3 The recovery rate range of β-lactoglobulin in the three matrices(n=18) β- A B (mg/kg ) β- mg/kg (%) β-*mg/kg (%) ~ ~ / ~ ~ / ~ ~ ~ ~ * : B 表 4 IFP 参考物质 ELISA 检测结果 Table 4 ELISA results of IFP reference materials A ELISA (mg/kg ) ElisaSystems R-biopharm B ( x, mg/kg) (SD, mg/kg) (CV, %) (%) , ( β- ) [11] 2008 (Major Allergen Declaration (MAD) Guidelines) β-,, β-, ELISA,, :, 参考文献 [1] Hosta, Halkensa. A prospective study of cow milk allergy in Danish infants during the first 3 years of life clinical course in relation clinical and immunological type of hypersensitivity reaction [J]. Allergy, 1990, 45: [2] Hosta, Bahnasl. Food hypersensitivity and adverse reactions:a practical guide for diagnosis and management [M]. New York: Marcel Dekker, 1999: [3] Brownlow S, Morais JH, Cooper R, et al. Bovine -lactoglobulin at 1.8 A resolution still an enigmatic lipocalin [J]. Structure, 1997, (5): [4],,. [J]., 2007, 28(2): [5],,. [J]. (), 2008, 29 (4):

5 4, : β- 299 [6] Matsumoto N, Okochi M, Matsushima M, et al. Peptide array-based analysis of the specific IgE and IgG4 in cow s milk allergens and its use in allergy evaluation [J]. Peptides, 2009, 30 (10): [7] Cerecedo I, Zamora J, Shreffler WG, et al. Mapping of the IgE and IgG4 sequential epitopes of milk allergens with a peptide microarray based immunoassay [J]. J Allergy Clin Immunol, 2008, 122(3): [8] Matsumoto N, Okochi M, Matsushima M, et al. Development of peptide arrays for detection of IgE-binding epitopes in cow's milk allergens [J]. J Biosci Bioeng, 2009, 107(3): [9] Okada H, Kaji N, Tokeshi M, et al. Analysis of potent milk allergen using highly sensitive protein separation chip [J]. Nanomed: Nanotechnol, Biology Med, 2009, 3(4): 351. [10] Spiked Samples for Allergen Determination (February 2006) [ DB/OL]. [11] Monaci L, Tregoat V, Hengel AJ. Milk allergens, their characteristics and their detection in food: A review [J]. Eur Food Res Technol, 2006, 223: ( 责任编辑 : 张宏梁 ) 作者简介张海英,,, zhanghy@126.com 张霞,,, zhangxia_75@126.com ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆ ,,,,,,, 2013,, tougao@chinafoodj.com

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