Microsoft Word 许迪莘-二校 4958彩-图4

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1 6 12 Vol. 6 No Journal of Food Safety and Quality Dec., 2015 许迪莘 1#, 代凤英 2#, 杨寅 1, 邓奕 1, 杜庆庆 1, 陈旭 1, 张远 1, 杜美红 (1., ; 2. ( ), ) 2* 摘要 : 目的, 方法,,, 结果 7.5%, 5%, 1% 10%; 1 1, 37 3 d, 70% 结论, 1 1 关键词 : ; ; ; ; Application of gelatin and casein as blocking agent and stabilizer in preparation of immunomagnetic beads XU Di-Xin 2#, DAI Feng-Ying 1#, YANG Yin 2, DENG Yi 2, DU Qing-Qing 2, CHEN Xu 2, ZHANG Yuan 2, DU Mei-Hong 1* (1. Beijing Scientific Instruments & Materials Corp, Beijing , China; 2. Beijing Center for Physical & Chemical Analysis (Beijing Engineering Research Center of Food Safety Analysis), Beijing , China) ABSTRACT: Objective To research the performance of the blocking agent and storage stable liquid of immunomagnetic beads, and to guarantee the capture efficiency of microorganism. Methods The gelatin and casein were used as blocking agent and preservation solution in the preparation of anti-salmonella immunomagnetic beads. The specific capture rate and shelf life of the immunomagnetic beads were obtained and examined. Results The non-specific binding rate of Escherichia coli was less than 5% after blocking the immunomagnetic beads with 7.5% of gelatin, the specific binding rate maintained above 70% after 3 d at 37 when the beads were preserved in 1 of casein and 1 of gelatin.. Conclusion As ablocking agent, gelatin 基金项目 : ( ) (2013YQ140371) (IG201307) (Z ) Fund: Supported by the Program of Beijing Academy of Science and Technology ( ), National Major Special Projectin Scientific Equipment (2013YQ140371), Innovation Team of Beijing Academy of Science and Technology (IG201307) and Beijing Science and Technology Program (Z ) # # XU Di-Xin and DAI Feng-Ying are co-first authors * 通讯作者 :,, dumeihong@beijinglab.com.cn *Corresponding author: DU Mei-Hong, Associate Researcher, Beijing Center for Physical & Chemical Analysis, Beijing Engineering Research Center of Food Safety Analysis, Beijing , China. dumeihong@beijinglab.com.cn

2 could reduce the non-specific binding of immunomagnetic beads, and 1 of gelatin with 1 of casein in the preservation solution exhibited a conducive ability to keep the activity of antibody on immunomagnetic beads. KEY WORDS: immunomagnetic beads; gelatin; casein; blocking agent; preservation solution 1 引言 [1],, ( ),,,,,, [2],, 9.72%,,,,,,,,,,, [3] [2] [4-7], [9-12] [13] [8],,, 2 材料与方法 2.1 材料 ( 180 nm, ); IgG ( KPL, IgG ); IgG (, IgG ); (BSA, Sigma-Aldrich ) 0.22 μm ; (Sigma-Aldrich ) 0.22 μm ; (Sigma-Aldrich ) 0.22 μm ; (Salmonella typhimurium, : ATCC14028); (Escherichia coli, : ATCC25922); (Shigella flexneri, : ATCC12022) IgG-FITC(, ); (FBS, Gibco ) 0.22 μm ; (10 mmol/l ph , ); ( ); 1-(3- )-3- (EDC) N- (NHS) (J&KCHEMICAL ); 2.2 仪器 (, : BSC-1300Ⅱ A2, ), (Thermo scientific, : IGS180, ), (SANYO, : MLS-3780, ), (OLYMPUS, : BX43/53, ), (Sartorius, : CPA225D, ), (Milli-Q, ) 2.3 方法 磁球封闭 2 ml, PBST 2 5 mg/ml EDC 5 mg/ml NHS, min, PBS 2, min, PBS 免疫磁球制备 2 ml, PBST 2 5 mg/ml EDC 5 mg/ml NHS, min, PBS 2 ( IgG ),, 37 3 h, PBS 2, min, PBS 免疫磁球对目标菌的捕获 10 2 cfu/ml 1 ml

3 12, : 4957,,, 30 min,, 1 ml PBS -, vortex, 36 ±1 18 h, y=b/(a+b) 100% (a b ) 免疫荧光实验 - 1% FBS ( IgG ), min 30 min, 1% FBS PBS 2 FITC IgG-FITC 30 min 30 min, 1%FBS PBS 5, 保存液的制备, 20% BSA 10% 1 1 ~5 1 ~ ~5 37, 37 1 d 2 d 3 d 7.5%, 5%,,,, [14],, 7.5% 1%, 7.5% 3 结果与分析 3.1 免疫磁球封闭液的优化 不同封闭液对磁球吸附效率的影响 7.5% 1% 0.1% 1% 0.1% 1% 0.1% 1mL 10 2 cfu/ml, , 1%, 9.73%±0.69%, 7.49%±0.14% 0.1% 1% 10% ph, 0.8%~1.2%, 1%, 7.5%, 4.72%±0.48%, 3.44%±0.90% 1% 0.1%, 10%, 1 Fig. 1 Adsorption rate of magnetic beads on E.coli prepared by different blocking agent 2 Fig. 2 Adsorption rate of magnetic beads on Sh. flexneri prepared by different blocking agent

4 不同封闭液对免疫磁球捕获效率的影响 7.5% 1% 1% - 1mL 10 2 cfu/ml, 3, 84.68%± 0.31%, 1%, 80.73%±0.76%, 80%,, 1% 7.5% 1%, 7.5% 1% 3.2 免疫荧光验证试验 3 Fig. 3 Specific capture rate of immunomagnetic beads prepared by differentblocking agent 3, 7.5% -, 84.57%±0.72%, 1% 7.5%, 7.5% 1% 1% -,, -,, ;, FITC, 4 A: 1% BSA, B: 1% BSA ; C: 7.5% Gelatin, D: 7.5% Gelatin ; E: 1%Casein, F: 1%Casein Fig. 4 A: 1% BSA blocked immunomagnetic beads, B: 1% BSA blocked immunomagnetic beads to capture bacteria, C: 7.5% Gelatin blocked immunomagnetic beads, D: 7.5% Gelatin blocked immunomagnetic beads to capture bacteria, E: 1%Casein blocked immunomagnetic beads, F: 1%Casein blocked immunomagnetic beads to capture bacteria

5 12, : A C E,, 7.5%, FITC,, B D F,, - 1%, 7.5%, IgG-FITC, [15],,, FITC, - 2.1, 1%, 7.5% 3.3 明胶 酪蛋白对免疫磁球保存液的影响,, [16,17],, 20, 1,, d 1 90% d 3, 20 5,, 20 ; 1, ; 1,,, 4 结论,,,, 7.5% - FITC, 1%BSA 7.5% , 37 3 d, 70%,,,, 表 1 保存液中明胶 吐温 20 酪蛋白含量和保存时间对捕获率的影响 Table 1 The effect of content of gelatin,twain 20 and casein in the preservation solution and storage time on the capture rate 20 (%) 1 d 2 d 3 d A ± ± ±2.27 B ± ± ±0.08 C ± ± ±2.96 D ± ± ±0.21 E ± ± ±0.78 F ± ± ±1.50 G ± ± ±0.60 H ± ± ±1.78 I ± ± ±0.03 J ± ± ±4.18

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