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1 5 7 Vol. 5 No Journal of Food Safety and Quality Jul., 2014 杨亚杰 1, 倪小琴 1, 王茜 2, 游琼 2, 张富生 2, 赖卫华 (1., ; 2., ) 1* 摘要 : 目的 方法,, EDC, NC, 结果 25 min, 100 ng/ml, BSA OVA, ng/ml, (89.03±5.2)% (93.47±6.9)% (91.2±7.8)%, (87.69±6.2)% (92.73±8.3)% (89.82±8.5)% 结论 关键词 : ; ; ; Fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk YANG Ya-Jie 1, NI Xiao-Qin 1, WANG Qian 2, YOU Qiong 2, ZHANG Fu-Sheng 2, LAI Wei-Hua 1* (1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang , China; 2. Jiangxi Agricultural Product Quality Safety and Inspection Center, Nanchang , China) ABSTRACT: Objective To develop a fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk. Methods Casein was used as the antigen to immunize the New Zealand rabbits to make the corresponding polyclonal antibody against the antigen, and EDC mediated method was used to couple the purified polyclonal antibody with fluorescent microspheres. The filter pad, sample pad, conjugate pad, NC membrane, and absorption pad were assembled to develop the fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk. Results The results could be obtained within 25 min without sophisticated equipment, and the limit of detection of the test strip was 100 ng/ml. The developed assay was highly specific to the casein protein as a result of that it had no cross-reactivity with other non-target proteins, such as BSA and OVA. The recovery rates of the intra-assay at casein concentrations of 100.0, 500.0, and ng/ml were (89.03±5.2)%, (93.47±6.9)% and (91.2±7.8)%, respectively, while the inter-assay were (87.69±6.2)%, (92.73±8.3)% and (89.82±8.5)%, respectively. Conclusion The fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk with the advantages of rapid, convenient, and high sensitivity was established preliminarily. KEY WORDS: casein; lateral flow assay; fluorescent microspheres; quantitative detection * 通讯作者 :,, talktolaiwh@163.com *Corresponding author: LAI Wei-Hua, Professor, State Key Laboratory of Food Science and Technology, Nanchang University, No.235, Nanjing East Road, Nanchang , China. talktolaiwh@163.com

2 7, : 引言, 20 8, (FAO) (WHO), [1], [2] 20% 1 [3], 3%~7.5% [4],, 2007/68/EC,,, [5] 3%, (casein) (lactoglobulin) (lactalbumin) [6], 80%,,,,,,, [7,8],,,, [9,10] [11, 12] [13] [14] [15,16] [17] [18-20],,,,,,, [21], [22] [23] O157:H7 [24],,,,, 2 材料与方法 2.1 材料和仪器 实验材料 ( 175 nm, 470 nm, 525 nm, COOH= 443 µeq/g), ; NC : Prima 40, Whatman ; CN 140 and CN 95, Sartorius Stedim Biotech ;, Millipore ; 1-(3- )-3- (EDC, 97.0%) (BSA, 100G) (OVA, 25G) (C G, C G) (500G), Sigma ; ( 0.5 mg/ml), ;, 主要仪器, ; XYZ3050, Biodot ; DZX-6090B, ;, 2.2 方法 抗原制备, [25,26], PBS(pH=9.0) PBS(pH=7.0) min (0.1%DTT, 6 mol/l ) PBS min, r/min 30 min,, 抗酪蛋白多克隆抗体制备及纯化 2.2.1, 6 (, 2.0 kg ), - [27] 荧光微球偶联抗体 EDC [28,29] : 3 ml ph mol/l PB 0.15 mg, 2.7 μl 5 mg/ml EDC, 0.5 mg/ml,

3 h, BSA( 1%) 30 min 9000 r/min 4 10 min,, 300 µl, ( ), 4 ELISA =( - )/ 荧光微球免疫层析试纸条的组装 Bio-Dot XYZ mg/ml 0.5 mg/ml NC (T ) (C ), NC 30 2 h NC, 3.9 mm ( 1 ) 检测方法, 4 μl μl, 3 min, 条件优化 NC NC, CN 140, CN 95, Prima 40; mg/ml, NC T ; μg, ;, -, 检测样品 100 mg 10 ml 0.01 mol/l PBS, 2.2.1, 4, 9000 r/min 15 min, 100 μl 4 μl -, 3 min, 评价实验 灵敏度评价 10 mg/ml, mg/ml,, FI T /FI C B 0 B, 特异性评价 1 mg/ml BSA OVA,, 100 μl, 1 μg/ml, 0.01 mol/l PBS, 25 min,, 重复性和稳定性评价, ( ng/ml),, 3 结果与分析 3.1 抗酪蛋白多克隆抗体制备及选择 ELISA,, 2, 6, 荧光微球 - 抗体复合物的偶联率 ELISA, Fig. 1 1 The structure of the test strip

4 7, : Fig. 2 Titers of pab generated by six kinds of caseins,, μg 94.6% 92.3% 98.7%, 90%, EDC, 3.3 条件优化 L 9 (3) 3 NC, 1 1, T, : Prima 40 NC, T 0.8 mg/ml, 1.0 μg 3.4 检测时间的确定,, 3, 30 min, T/C,, 25 min 3.5 灵敏度实验结果, 4 B/B 0 3 SD, 100 ng/ml 3.6 特异性实验结果 PBS 1 mg/ml OVA BSA 1 μg/ml, 5, OVA BSA T/C,, OVA BSA Table 1 表 1 正交试验分析结果 The analysis results of the orthogonal test No. NC T (mg/ml) (μg) T ( ) (%) 1 CN CN CN CN CN CN Prima Prima Prima * : 1 ng/ml

5 Fig. 3 3 The detection time of the strip 5 Fig. 5 The specificity of the strip 3.7 重复性和稳定性结果,, 2, 5.2%~7.8% 6.2%~8.5%, 89.03%~91.2% 87.69%~92.73% 4 结论 4 Fig. 4 Dose-response curves of the strip,, 100 ng/ml, 25 min,, Table 2 表 2 重复性和稳定性结果 (n=3) The stability and precision of the strip (n=3) (ng/ml) (%) (%) (%) (%) 参考文献 [1] Luo YK, Bu GH. Cow s Milk Allergens and Technologies to Control Allergenicity[M]//Multidisciplinary Approaches to Allergies[J]. Springer Berlin Heidelberg, 2012: [2] Monaci L, Tregoat V, Hengel AJ, et al. Milk allergens, their characteristics and their detection in food:a review [J]. Eur Food Res Technol, 2006, 223: [3] Host A, Jacobsen HP, Halken S, et al. The natural history of cow's milk allergy/intolerance [J]. Eur J Clin Nutr, 1995, 49(Suppl. 1): [4],,.

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