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1 6 9 Vol. 6 No Journal of Food Safety and Quality Sep., , 李亚楠, 王瑞, 孙元媛, 杨典原, 黄种乾 2, 王云贵 3, 李涛 4, 黄登宇 (1., ; 2., ; 3., ; 4., ) 1,2* 摘要 : 目的 1- (ELISA), 方法,,,, 结果 Y= X (r 2 =0.9922), 0.177~ ng/ml, IC ng/ml; μg/kg, 82.6%~104.7%, 3.6%~9.3%, 4.3%~12.4% 结论, 关键词 : ; ; Determination of nitrofurantion metabolite by direct competitive enzyme-linked immunosorbent assay LI Ya-Nan 1, 2, WANG Rui 1, 2, SUN Yuan-Yuan 1, 2, YANG Dian-Yuan 1, 2, HUANG Zhong-Qian 1, 2, WANG Yun-Gui 3, LI Tao 4, HUANG Deng-Yu 1,2* (1. College of Life Science, Shanxi University, Taiyuan , China; 2. The Food and Drug Safety Rapid Inspection Center, Shanxi University, Taiyuan , China; 3. Shenzhen Bioeasy Biotechnologies Co., Ltd., Shenzhen , China; 4. Shanxi Vanguard Technology Co., Ltd., Taiyuan , China) ABSTRACT: Objective To develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the detection of nitrofurantion metabolite in animal tissues. Methods The horseradish peroxidase labeled antigen was prepared by activated ester method. The dilutions of the enzyme labeled antigen and monoclonal antibody were determined by chequerboard titration, and the reaction conditions were optimized to establish the direct competitive enzyme-linked immunoassay for the detection of nitrofurantion metabolite. Then the assay was evaluated. Results The regression equation was Y= X (r 2 =0.9922) with a linear detection ranging from to ng/ml. The IC 50 was ng/ml. The limit of detection (LOD) in pork, chicken and fish samples was 0.115, and μg/kg, respectively. Recoveries were between 82.6%~104.7% when AHD was spiked to samples. The coefficient of variation of intra-and inter-assay was 3.6%~9.3% and 4.3%~12.4%. Conclusion The ELISA method is fast and accurate, and suitable for the detection of nitrofurantion metabolite residue in animal tissues. KEY WORDS: nitrofurantoin metabolite; direct competitive enzyme-linked immunosorbent assay; animal tissues * 通讯作者 :,, Huangdy1110@126.com *Corresponding author: HUANG Deng-Yu, Associate Professor, Shanxi University, No.92, Wucheng Road, Xiaodian District, Taiyuan , China. Huangdy1110@126.com

2 引言,,, [1],, [2-4], 1-- (1-amino-hydantoin, AHD) (3-amino-5-morpolinomethyl-2-oxazolidone, AMOZ) 3--2-(3-amino-2-oxazolidinone, AOZ)(semicarbazide, SEM) [5] [6], [7,8], AHD [9], AHD [6,9-12] [13-15] [16],, ;, ;,,, ELISA, 2 材料与方法 2.1 材料与仪器 1-(AHD) 1-(2-)- (NPAHD)(HRP) N,N- (DMF) N-(NHS) N,N- (DCC) (TMB) (BSA) ( Sigma ); AHD (); 1-(4- )- (CPAHD)(); 30% H 2 O 2 (4-CBA) (); Na 2 HPO 4 12H 2 O K 2 HPO 4 NaOH ( ) TMB : 10.0 mg TMB 5 ml, 0.5 ml 20 ml - (ph 5.0, 0.05 mol/l), 10 μl 30% H 2 O 2,, BSA224S ( Sartorius ); INFINITE 200PRO ( Tecan ); UPT-I-10T (); BSD-100 ( ); SC-3610 ( ); 96 (Thermo Fisher Sicentific ); (Solarbio ) 2.2 实验方法制备酶标抗原 (CPAHD-HRP) AHD,, [17] CPAHD HRP [18], : 8.4 mg CPAHD 0.36 ml DMF, 6.3 mg NHS 11.4 mg DCC,, 14 h, A ; 7.5 mg HRP 3.0 ml (PBS, ph 7.4, 0.01 mol/l), B ; A B, 2 h, 4 (12 h), PBS 96 h, 4500 r/min 5 min,, CPAHD-HRP,, AHD 直接竞争 ELISA 检测步骤 AHD (CBS, ph 9.6, 0.05 mol/l) 96, 100 μl/well, ;, PBST ( 0.05% Tween-20, ph 7.4, 0.01 mol/l) 3,,, 200 μl/well, 37 2 h, ; 50 μl NPAHD 50 μl,, ; TMB, 100 μl/well, 37 ; 2 mol/l H 2 SO 4, 50 μl/well,, 450 nm OD

3 9, : 优化反应条件 (1) OD 1 1: : : :8000 1:16000, 1:10 1:20 1:40 1:80 1:160 1:320, OD (2) 4 (1 ) 37 2 h 4 (2 ) 37 2 h(3 )3, 1%(1 ) 1%(2 ) 1% BSA(3 )3, 40 min 1 h 2 h 2.5 h 4, 10 min 20 min 30 min 3, 3, ELISA, IC 50 OD max /IC 50, 方法学评价 (1), NPAHD NPAHD ng/ml, 2.2.2, OD, 3 A/A 0 (A NPAHD OD, A 0 NPAHD OD ), NPAHD,,, IC 50 (IC 20 ~IC 80 ) (2) NPAHD AHD AMOZ AOZ SEM NPAMOZ NPAOZ NPSEM 4-CBA ng/ml, 2.2.2, (CR) (3), ng/g 3 AHD 5, 5, [19], : 20 g,, 2.0 g 3, 9 ml,, ng/g AHD, ; 1.00 mol/l HCl 1.0 ml 0.01 mol/l 200 μl,, h;, 0.10 mol/l K 2 HPO ml,, 1.00 mol/l NaOH 0.8 ml,, 10.0 ml, 1 min, 4500 r/min 10 min,, ; 3.0 ml, 1.0 ml PBS, 4500 r/min 10 min, (4) 20,, AHD ELISA, A/A (LOD) 3 结果与分析 3.1 酶标抗原 CPAHD-HRP 的紫外鉴定 CPAHD 299 nm, HRP 403 nm, CPAHD-HRP 308 nm 406 nm CPAHD-HRP CPAHD HRP, CPAHD HRP,, CPAHD-HRP 1 CPAHD HRP CPAHD-HRP Fig. 1 The UV spectrum of CPAHD, HRP and CPAHD-HRP 3.2 反应条件的优化抗体与酶标抗原稀释度的优化 OD 1,, OD

4 Table 1 表 1 不同抗体和酶标抗原稀释度下 OD 值 (n=3) The OD values of different dilution of antibody and enzyme-labeled antigen (n=3) 1:1000 1:2000 1:4000 1:8000 1: : ± ± ± ± ± : ± ± ± ± ± : ± ± ± ± ± : ± ± ± ± ± : ± ± ± ± ± : ± ± ± ± ±0.0082, 1:4000, 1:80, OD 包被条件的优化, IC 50 OD max /IC 50, OD max /IC 50,, 4 3 AHD ELISA Fig. 3 The optimization of blocking liquid for ELISA of AHD 竞争时间的优化 IC 50 OD max /IC 50 4,, IC 50, 1 h, IC 50 2 h, OD max /IC 50, 1 h 2 AHD ELISA Fig. 2 The optimization of coating condition for ELISA of AHD 封闭液的优化 IC 50 OD max /IC 50 3, 3, IC 50, 3 OD max /IC 50,, 1%BSA Fig. 4 4 AHD ELISA The optimization of competitive reaction time for ELISA of AHD

5 9, : 显色时间的优化 IC 50 OD max /IC 50 5,, IC 50, 20 min, OD max /IC 50, 20 min 0.1%,, AHD, 表 2 AHD ELISA 法特异性测定结果 (n=3) Table 2 The specificity of AHD by ELISA(n=3) IC 50 /(ng/ml) CR/(%) NPAHD >1000 <0.1 >1000 <0.1 5 AHD ELISA Fig. 5 The optimization of developing time for ELISA of AHD >1000 <0.1 NPAOZ >1000 <0.1 NPAMOZ >1000 < 方法学评价标准曲线的建立, 1:4000, 4, 1%BSA, 1:80, 1 h, 20 min, AHD ELISA,, ~ ng/ml, Y= X (r 2 =0.9922), IC ng/ml 6 AHD ELISA Fig. 6 Standard curve of AHD by ELISA 特异性测定 2, AHD ELISA 38.1%, NPSEM >1000 <0.1 AHD >1000 <0.1 AMOZ >1000 <0.1 AOZ >1000 <0.1 SEM >1000 <0.1 4-CBA >1000 <0.1 >1000 < 准确性和精密度,, AHD ELISA, 3, 82.6%~104.7%, 3.6%~9.3%, 4.3%~12.4%, 样品检测限, AHD ELISA 3, μg/kg

6 表 3 AHD ELISA 法的准确性和精密度 (n=5) Table 3 The accuracy and precision of AHD by ELISA (n=5) /(ng/g) /(%) CV/(%) CV/(%) 表 4 AHD ELISA 法的样品检测限 (n=20) Table 4 The sample detection limit of AHD by ELISA (n=20) 4 结论 /(μg/kg) ,,,, 38.1%, 0.1% 3 3,,,,,, 参考文献 [1],.[J]., 2013, 4(1): Gao Jie, Miao Hong. Recent development of analytical methods for veterinary drug residues [J]. J Food Saf Qual, 2013, 4(1): [2] Vass M, Hruska K, Franek M. Nitrofuran antibiotics: a review on the application, prohibition and residual analysis [J]. Vet Med (Praha), 2008, 53: [3] O keeffe M, Conneely A, Cooper KM, et al. Nitrofuran antibiotic residues in pork: the Food BRAND retail survey [J]. Anal Chim Acta, 2004, 520: [4] Cheng C, Hsieh K, Lei Y, et al. Development and residue screening of the furazolidone metabolite, 3-amino-2-oxazolidinone (AOZ), in cultured fish by an enzyme-linked immunosorbent assay[j]. J Agric Food Chem, 2009, 57(13): [5] Auro A, Sumano H, Ocampo L, et al. Evaluation of the carcinogenic effects of furazolidone and its metabolites in two fish species [J]. Pharmacogen J, 2004, 4: [6] Dongkyum K, Byungjoo K, Seok-Won K, et al. An optimized method for the accurate determination of nitrofurans in chicken meat using isotope dilution-liquid chromatography/mass spectrometry [J]. J Food Compos Anal, 2015, 40: [7] Commission Regulation (EC). No.1442/95 Amending decision as regards the setting of minimum required performance limits for certin residues in food of animal origin [Z]. OJEC, 1995, L143: 26. [8] Federal Register. Topical nitrofurans; extralabel animal drug use; order of prohibition [R]. Fed Regist, 2002, 67: [9] Noelia VT, Patricia PB, Roberto RG, et al. Determination of nitrofurans metabolites in seafood by ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry [J]. J Food Compos Anal, 2013, 30: [10] SN/T [S].

7 9, : 3729 SN/T Determination of metabolites of nitrofurans residues in animal origin food for import and export HPLC-MS/MS [S]. [11] GB/T [S]. GB/T Method for the determination residues of the metabolites of nitrofuran in pork, beef, chicken, porcine liver and aquatic products LC-MS-MS method [S]. [12] GB/T / [S]. GB/T Determination of residues of nitrofuran metabolites in foodstuffs of animal origin HPLC-MS/MS method [S]. [13] Jiang WX, Luo PJ, Wang X, et al. Development of an enzyme-linked immunosorbent assay for the detection of nitrofurantoin metabolite, 1-amino-hydantoin, in animal tissues [J]. Food Control, 2012, 23: [14] SN/T [S]. SN/T Determination of residues of nitrofuran metabolites in foodstuffs of animal origin for export ELISA method [S]. [15],,. ELISA 1- [J]., 2008, 29(8): Xu YP, Jin ZY, Xu CL. Development of a sensitive ELISA method for detection of 1-aminohydantoin, a tissue-bound metabolite of the nitrofurantoin [J]. Sci Technol Food Ind, 2008, 29(8): [16],,,. [J]., 2013, 25(1): Liu AC, Liu C, Zhao Y, et al. Rapid determination of nitrofuran metabolites in aquatic products by colloidal gold method [J]. Acta Agric Zhejiangsis, 2013, 25(1): [17]. [D]. :, Zheng LL. Study of enzyme-linked immunosorbent assay for the detection of furaltadone and furantoin [D]. Tianjin: Tianjin University of Science and Technology, [18]. AHD [D]. :, Shi JL. Study on the rapid detection technology of the metabolite AHD of nitrofurantoin [D]. Beijing: Chinese Academy of Agricultural Sciences, [19],,,. [J]. ( ). Lv YX, Wang R, Huang DY, et al. Development of a direct competitive chemiluminescent enzyme immunoassay for detection of furaltadone metabolite [J]. Sci Technol Food Ind (Accepted). 作者简介 ( 责任编辑 : 杨翠娜 ) 李亚楠, 硕士研究生, 主要研究方向为食品新工艺与食品安全 nan4050@163.com 黄登宇, 博士, 副教授, 硕士生导师, 主要研究方向为食品卫生检测 Huangdy1110@126.com

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<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6> 227 ELISA 刘沙洲 1,2, 桑小雪 1, 欧阳华学 3, 雷绍荣 3, 白林含 1, * (1. 四川大学生命科学学院, 四川成都 610065;2. 成都市食品药品检测中心, 四川成都 610045; 3. 四川省农业科学院分析测试中心, 四川成都 610066) 目的 : 比较