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1 7 3 Vol. 7 No Journal of Food Safety and Quality Mar., 2016 * 郑琦, 曹立民, 郑洪伟, 林洪, 隋建新 (, ) 摘要 : 目的 方法 A, sepharose 4B, ELISA ;, (HPLC) ; HPLC 结果 5.67, L/mol; 4 6 h ; HPLC 0.2 mol/l ( 0.5 mol/l NaCl, ph 2.3), %±0.03%(n=6); 5, 85% ; 0.02%NaN 3 PBS, 110 d 800 ng 75% ; μg /kg, 63.29%±5.30% 80.15%±6.27% 83.87%±0.88%; 2~4 h 结论, 关键词 : ; ; Preparation and characterization of an immunoaffinity column for the selective extraction of neomycin ZHENG Qi, CAO Li-Min, ZHENG Hong-Wei, LIN Hong, SUI Jian-Xin * (College of Food Science and Engineering, Ocean University of China, Qingdao , China) ABSTRACT: Objective To prepare and characterize an immune affinity column for the selective extraction of neomycin. Methods The immune affinity column of neomycin (NEO) was prepared by coupling CNBr-activated Sepharose-4B with the purified anti-neo polyclonal antibody. An indirect competitive ELISA method was established to detect the specificity, affinity constant and titer of antibody. The changes of protein concentrations were determined by Coomassie brilliant blue method. HPLC method was used to explore the optimal elute condition. A rapid and reliable immune affinity column (IAC) clean-up based high performance liquid chromatography (HPLC) method was developed for the determination of neomycin (NEO) in Penaeus vannamei Boone products. Results The titer of the lgg was improved 5.67 times than before and the affinity constant (Ka) of lgg was about L/mol. The optimal coupling conditions were at 4 coupling 6 h. The immunoaffinity column could be eluted with 5 ml 0.2 mol/l Citrate buffer (0.5 mol/l NaCl, ph 2.3). The average recoveries were %±0.03% (n=6) and the rate of recovery of neomycin was more than 85% after 基金项目 : (2014ZZCX02703) Fund: Supported by Special Fund for Shandong Independent Innovation and Achievements Transformation (2014ZZCX02703) * 通讯作者 :,,, suijianxin@ouc.edu.cn *Corresponding author: SUI Jian-Xin, Associated Professor, College of Food Science and Engineering, Ocean University of China, No.5, Yushan Road, Shinan Distract, Qingdao , China. suijianxin@ouc.edu.cn

2 repeatedly for 5 times. After storage at 4 for 110 d, the rate of recovery to neomycin was more than 75% after loaded NEO for 800 ng. The average recoveries of NEO in spiked Penaeus vannamei Boone under levels of 500, 5000 and 7500 μg/kg ranged from 63.29% to 83.87% with the relative standard deviations of 0.88%~6.27%. Compared with solid phase extraction, immunoaffinity extraction saved 2~4 hours. Conclusion The preparation neomycin affinity column has the advantages of high accuracy and good reproducibility, which has the potential application value in food safety testing. KEY WORDS: neomycin; immunoaffinity column; high performance liquid chromatography 1 引言 2-,,,, [1], No 1056/2013, μg/kg [2], μg/kg,, ( ) [3-16] [3-5] [6],,,, [3,4], ; [7-9],,, [8,9] ; [7] ; [10-14],, ; HLB [15] - [16],,, : (1) ; ; (2) ;,,,, 2~4 h; (3) ;,, (LC-MS/MS) [17,18], ;, [3,5,16],,, [19-21],,,,, 2 材料与方法 2.1 仪器 试剂与材料 Agilent 1260 (, Aglient ); MuLtiskan MK3 ( Labsystems ); A ( GE Healthcare ); CNBr-activedSepharose 4B( GE Healthcare ); ( )

3 3, : 1039 ( ) HSA(Morton Grore ) (TEDIA) (TEDIA) Fmoc-Cl(Fluka) () (Sigma ); ; 2.2 实验方法 多克隆抗体性能鉴定 , A,, SDS-PAGE , (NEO-BSA) 1 μg/ml 1: : 5000 lgg, 0.8~1.0 (NEO-BSA) 1 μg/ml 1:5000, lgg, (PBS) A lgg, ELISA, 3, NEO-BSA 1 μg/ml, 4 2 (10~6400 ng/ml),, 50 μl, 1:25000, 50 μl, 3 OD 450 B 0, OD 450 B B/B 0 (%), B/B 0 (%), 4, 50% IC 50, [22] ELISA, NEO-BSA μg/ml, 15 μg/ml (),, (n-1) K 2(n[Ab] -[Ab] t ( L/mol) [Ab] [Ab] t Ag Ag t, OD 50 (mol/l), n=ag/ag t 免疫亲和柱的制备 0.15 g sepharose 4B, 4 25, h lgg, G-250 [23],, 6, 免疫亲和柱的性能评价 ) : (0.01 mol/l PBS, ph 7.4) 1 ml/min 5 min 2) : 2 mg/ml, 4, 1 (0.01 mol/l PBS, ph 7.4) 400 ng/ml, 2 ml,, 3) : 10 ml 1 ml/min 4) : Kandimalla [25], ph 3, 0.2 mol/l ( 0.5 mol/l NaCl, ph 2.3) 0.2 mol/l ( 0.5 mol/l NaCl, ph 2.7) 0.2 mol/l ( 0.5 mol/l NaCl, ph 2.7) 0.1 mol/l (ph 2.7) 0.2 mol/l ( 0.5 mol/l NaCl, 1% DMSO, ph 2.3) ph (80:20, V:V) - (85:15, V:V) ph mol/l 7:3 6:4 5:5

4 1040 7, 1 ml/min, 5 ml (n=3); [24],,, 400 μl 0.5 mol/l (ph8.0) 400 μl 80: mol/l HSA 100 μl 100 μl ( g Fmoc-Cl 5 ml ),, 15 min, 60 μl 0.1 mol/l,,, : : Agilent Clipse Plus-C18 (5 μm, mm); : - (90:10, V:V); : 1.0 ml/min; : =260 nm, =315 nm; 25 ; : 20 μl 5) : (0.01 mol/l PBS, ph7.4),, ml 100 ng/ml PBS 1 ml/min, 10 ml, 5 ml, HPLC,, 洗脱液中新霉素浓度 ( ng / ml) 体积 ( ml) 柱容量 ( ng/ml) 亲和柱柱床体积 ( ml) , , 5, HPLC, d,, HPLC, 800 ng,, HPLC 对虾肉样品加标回收率的测定 (0.01 mol/l PBS, ph 7.4) 2 mg/ml ng/ml, 2 ml ( μg) 2.0 g ( HPLC ), 30 min; 8 ml ( 1.36 g KH 2 PO 4, 800 ml, 1 mol/l HCl ph 4.0, 0.15 g EDTA-2Na 100 g, 1000 ml) 5 min, r/min 10 min, 50 ml, 10 ml 5 min, r/min 10 min,, 10 ml 10 ml 5 min, r/min 5 min, ;, 6 PBS, ph 7.4; 1 ml/min, 10 ml 5 ml, 2 mol/l Tris ph7.4, HPLC, (NEO Tris ) 3 结果 3.1 多克隆抗体的性能鉴定 抗体的纯化 A, SDS-PAGE ( 1),, lgg, 55 kd;, 25 kd,, 1 SDS-PAGE Fig. 1 Results of SDS-PAGE of anti-neo polyclonal before and after the purification A: Marker; B: ; C: A ( ); D: A: Marker; B: polyclonal antibody before purification; C: Impurity protein; D: polyclonal antibody after purification

5 3, : 抗体效价的测定 2, lgg 80, OD , lgg 15 lgg 85, 2.1, A 5.67, 3 NEO (n=3) Fig. 3 Relative affinity curve of polyclonal antibody (n=3) Fig. 2 2 (n=3) Efficacy of specific unpurified and purified anti-neo polyclonal and negative control (n=3) 3.2 免疫亲和柱制备 0.15 g CNBr-sepharose 4B, 4 25, h, 4, 6 h,, 4 6 h 抗体特异性的测定 1, <0.01%;, 表 1 抗血清与其他药物的交叉反应率 (n=3) Table 1 Cross reactivity of antiserum with other medicines (n=3) (%) <4.30 <0.01 <0.01 < 抗体亲和常数的测定 S ( 3) n=2, 3 K Ka= L/mol, >10 8 L/mol, lgg 4 (n=3) Fig. 4 Effects of time and temperature on ligand coupling (n=3) 0.15 g sepharose-4b 0.5 ml, lgg mg, 2182 ng/ml 6, 91.86%±7.05%,

6 %,, 3.3 免疫亲和柱性能评价 HPLC 筛选亲和柱洗脱剂, Kandimalla [25], ph 3, 800 ng, 5 ml,, 2,,, ;,,, ph,, ph,,,,,, ph,, 0.2 mol/l ( 0.5 mol/l NaCl, ph 2.3) 亲和柱的稳定性 HPLC, 2182 ng/ml, 5, 85% 0.02%NaN 3 4, 800 ng, 110 d, 75.44%( 5);,,, 5 (n=3) Fig. 5 Stability of immunoaffinity column(n=3) Table 2 表 2 不同洗脱液对于亲和柱洗脱效果的影响 (n=3) Effects of different buffers on affinity column elution (n=3) (%) (%) 0.2 mol/l ( 0.5 mol/l NaCl, ph 2.3) mol/l ( 0.5 mol/l NaCl, ph 2.7) mol/l ( 0.5 mol/l NaCl, ph 2.7) mol/l (ph 2.7) mol/l ( 0.5 mol/l NaCl, 1% DMSO, ph 2.3) (80:20, V:V), ph (85:15, V:V), ph mol/l - (70:30, V:V), ph mol/l - (60:40, V:V), ph mol/l - (50:50, V:V), ph

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9 3, : 1045 antigens from antibodies [J]. Biosens.Bioelectron, 2004, 20(4): [26],,. [J]., 2014, 8(1): Zhao LY, Hao AY, Liu YH. Study on repeated usings of aflatoxins immunoaffinity columns [J]. Chin J Mod Drug Appl, 2014, 8(1): [27],,,. A [J]., 2009, (4): Hu GL, Zhao Y, Wang ZP, et al. Study on detection and repeated usings of ochratoxin A immunoaffinity columns in barley [J]. Chin J Food Technol Ind, 2009, (4): [J]., 2008, 29(4): Qiao H, Zhao LJ, Zhang Y, et al. Preparation of immunoaffinity column of chloramphenicol for trace analysis of chloramphenicol in milk [J]. J Food Res Dev, 2008, 29(4): 作者简介 ( 责任编辑 : 金延秋 ) 郑琦, 硕士, 主要研究方向为食品安全与质量控制 sdqingdaozhengqi@163.com [28],,,. [J]., 2003, 20(1): Liu CW, Zhai GS, Mai GX, et al. Principle and progress of solid phase extraction technology [J]. Agric Environ Dev, 2003, 20(1): 隋建新, 博士, 副教授, 主要研究方向为水产品安全与质量控制 suijianxin@ouc.edu.cn [29],,,.