3 4 Vol. 3 No. 4 2012 8 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., 300461; 2., 300457) 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 300456, China; 2. College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, 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 : : (10-46-1) * 通 讯 作 者 :,,, Email: zhangxia_75@126.com
296 3, - :, (ELISA); DNA [6-9] ELISA, PCR,,, PCR ELISA, β- ELISA 2 材 料 与 方 法 2.1 材 料 2.1.1 检 测 试 剂 盒 A B β- ELISA, ElisaSystems R-biopharm 2.1.2 阳 性 添 加 物 () β- (L-8005, Sigma ); β-( IFP ) 2.1.3 食 品 基 质 β- ; ( MPBio ) (sigma ) α- (sigma ); 2.2 方 法 2.2.1 技 术 细 节 比 较 2.2.2 准 确 度 试 验 2.2.2.1 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- 2.2.2.2 ELISA 检 测, : (%)=/ 100%, A B β- 0.10 5 mg/kg,, 5 10 150 200 mg/kg ; β- 0.1 1 5 10 mg/kg 2 3 2.2.3 精 密 度 试 验 (CV%) β-, 5 12.5 mg/kg,, 3, (CV%) CV%: CV(%)= SD/X 100%, SD, X 2.2.4 特 异 性 试 验 α- 100 mg/kg 3 结 果 3.1 技 术 细 节 比 较 A B, 1
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,, (, ), () 60 60 30 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, 0.50 0 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- 0.10 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 5 12.5 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),
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 (%) 5.0 0.16 0.14~0.17 87.5~106.2 5 / 10.0 0.32 0.27~0.31 84.3~96.9 5 / 150.0 4.8 4.2~4.9 87.5~102.1 5 / 200.0 9.6 9.1~9.4 94.7~97.9 10.8 112.5 * : 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 (%) 0.10 0.10 0.09~0.11 90.0~110.0 5 / 1.00 1.00 0.82~0.93 82.0~93.0 5 / 5.00 5.00 4.61~4.93 92.2~98.6 5.6 112 10.0 10.0 8.76~9.68 87.6~96.8 86.1 86.1 * : 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 5.00 12.50 5.00 12.50 1 4.52 10.61 6.02 14.62 2 4.53 10.83 5.98 13.98 3 4.52 10.55 5.84 13.89 1 5.25 9.89 5.36 15.21 B 2 5.31 9.93 5.64 14.68 3 5.22 9.97 5.45 15.46 ( x, mg/kg) 4.89 10.29 5.72 14.64 (SD, mg/kg) 0.40 0.41 0.67 0.63 (CV, %) 8.18 3.98 11.7 4.30 (%) 97.8 82.3 114.4 117.1 2007, ( 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: 587 596. [2] Hosta, Bahnasl. Food hypersensitivity and adverse reactions:a practical guide for diagnosis and management [M]. New York: Marcel Dekker, 1999: 99 111. [3] Brownlow S, Morais JH, Cooper R, et al. Bovine -lactoglobulin at 1.8 A resolution still an enigmatic lipocalin [J]. Structure, 1997, (5): 481 495. [4],,. [J]., 2007, 28(2): 346 348. [5],,. [J]. (), 2008, 29 (4): 43 45.
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): 1840 1847. [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): 589 594. [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): 324 330. [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]. www.r-biopharm.com [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: 149 179. ( 责 任 编 辑 : 张 宏 梁 ) 作 者 简 介 张 海 英,,, E-mail: zhanghy@126.com 张 霞,,, E-mail: zhangxia_75@126.com ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆ 2010 1,,,,,,, 2013,, Email : www.chinafoodj.com Email: tougao@chinafoodj.com