氟喹诺酮类药物多残留酶联免疫检测方法的建立

doi:10.3864/j.issn.0578-1752.2014.23.017

摘要/Abstract

摘要： 【目的】氟喹诺酮类药物（FQs）在畜牧业中广泛用于预防和治疗细菌性疾病，不合理使用导致在动物性食品中残留，严重危害消费者健康，其检测受到世界各国重视。制备抗多种FQs单克隆抗体（mAbs），建立间接竞争ELISA（icELISA）检测方法，为动物性食品中FQs多残留检测奠定基础。【方法】环丙沙星（CPFX）羧基上引入氨基丁酸后为半抗原（CPFX-A），质谱鉴定；分别用碳二亚胺法（DDC）和混合酸干法将半抗原与牛血清白蛋白（BSA）和卵清蛋白（OVA）偶联合成免疫原（CPFX-A-BSA）和包被原（CPFX-A-OVA），紫外和红外光谱鉴定是否偶联成功；CPFX-A-BSA 免疫Balb/c小鼠，间接ELISA和icELISA方法测定多抗血清效价和敏感性，选取抗血清效价高和敏感性好的鼠用于细胞融合；NS0细胞和脾细胞按1﹕5比例在PEG-1500作用下融合，筛选抗FQs杂交瘤细胞株，并进行效价、亚型、敏感性和交叉反应率鉴定；体内诱生腹水法制备mAb，液体石蜡作为诱导剂，每只鼠注射10⁸个杂交瘤细胞；选敏感性和广谱特异性好的腹水mAb，建立icELISA标准曲线，对icELISA检测方法优化，在鸡肉中添加10种FQs进行测定，将测定结果与HPLC法比较，用SPSS 17.0软件进行显著差异性分析。【结果】半抗原改造和人工抗原合成成功；3只鼠血清效价均达到1﹕1.28×10⁴以上，2号鼠血清效价最高（1﹕2.56×10⁴），且IC₅₀值最低（12.92 ng·mL^-1），选择2号鼠作为细胞融合用鼠；4次亚克隆筛选并鉴定后获得3株敏感广谱特异的杂交瘤细胞，分别命名为2H5、3D11、4F4，细胞上清效价分别为1﹕1 600、1﹕1 600和1﹕800，腹水效价分别为1﹕1.6×10⁶、1﹕8.2×10⁵和1﹕8.2×10⁵；icELISA方法的包被原浓度为1 μg·mL^-1，5%猪阴性血清4℃包被过夜，单抗1﹕40 000稀释，山羊抗鼠酶标二抗（GaMIgG-HRP） 1﹕8 000稀释；反应温度均为37℃，标准品与单抗同时加入反应15 min，洗板后加二抗反应25 min；显色10 min，2H5细胞株腹水敏感性和广谱特异性最好，2H5的线性回归方程为y=-28.022x+56.219，R²=0.9 782，对10种FQs的IC₅₀值分别为环丙沙星（CPFX）1.67 ng·mL^-1、诺氟沙星（NOR）1.82 ng·mL^-1、培氟沙星（PEF）1.97 ng·mL^-1、恩诺沙星（ENR）1.54 ng·mL^-1、单诺沙星（DAN）2.79 ng·mL^-1、洛美沙星（LOM）3.38 ng·mL^-1、氧氟沙星（OFL）5.50 ng·mL^-1、麻保沙星（MAR）4.40 ng·mL^-1、沙拉沙星（SAR）11.76 ng·mL^-1、二氟沙星（DIF）13.60 ng·mL^-1，与10种FQs的交叉反应率为12.3%—108.4%，与非FQs交叉反应率均低于0.01%，对10种FQs的检测限（LODs）为0.09 ng·mL^-1—0.64 ng·mL^-1。icELISA法鸡肉中10种FQs的回收率为80.5%—91.8%，HPLC法的回收率为85.1%—95.7%，二者变异系数均低于10.0%；两种检测方法差异不显著（P＞0.05）。【结论】该试验获得了高效价、敏感、广谱特异性的mAbs，建立的icELISA方法可用于同时检测鸡肉中10种FQs。

关键词: 环丙沙星, 氟喹诺酮类药物, 杂交瘤细胞株, 广谱特异性单克隆抗体, 间接竞争ELISA

Abstract: 【Objective】Fluoroquinolones(FQs) are widely used in veterinary medicine for the treatment and prevention of bacterial infection. With the increasing use, FQs residues in animal edible tissues have caused serious public health problems and attracted serious attention by research scholars all over the world. The objective of this study wasto produce class-specific monoclonal antibodies (mAbs) against fluoroquinolones (FQs), establish competitive indirect enzyme linked immunesorbent assay (icELISA), and in order to lay a foundation for detection of multi-residue FQs in animal foods.【Method】 The aminobutyric acid was introduced to carboxyl of ciprofloxacin as hapten (CPFX-A) and was proved by (+) ESI-MS spectrum, which was conjugated to bovine serum albumin (BSA) as immunogen (CPFX-A-BSA) by the N,N'-Dicyclohexylcarbodiimide (DDC) method, and to ovalbumin (OVA) as coating antigen(CPFX-A-OVA) by mixed anhydride method, respectively, which were then identified by infrared ray (IR) and ultraviolet (UV). Balb/c mice immunized by CPFX-A-BSA were selected for cell fusion, which was identified by ELISA and icELISA. Under the effect of PEG-1500, NS0 cells and spleen cells were fused at the ratio of 1﹕5. Hybridoma lines that secrete mAb against FQs were selected and their immunological traits were characterized by titer, subtype, sensitivity and cross reaction rate, which ascites were carried out by injecting 10⁸ hybridoma cells in vivo, and icELISA standard curve was established and optimized. High titer, class-specific monoclonal antibody was used to detect 10 FQs in chicken for calculating recovery rate and variation coefficient. The data were also compared with that of HPLC, and SPSS 17.0 software was used to conduct the significant difference analysis.【Result】 The hapten and artificial antigen were synthesized successfully and antiserum titers of three mice were higher than 1﹕1.28×10⁴, in which the titer and IC₅₀ of No.2 mouse were1﹕2.56×10⁴and 12.92 ng·mL^-1. Three hybridoma cell lines named 2H5, 3D11, and 4F4 were screened after 4 times subclone, which titers were 1﹕1 600, 1﹕1 600, and 1﹕800 in supernatants and 1﹕1.6×10⁶, 1﹕8.2×10⁵, and 1﹕8.2×10⁵ in ascites, respectively. The icELISA procedure was optimized at a concentration of CPFX- A-OVA for 1 μg·mL^-1 at 4℃ package overnight by 5% negative serum of pig, monoclonal antibody and GaMIgG-HRP were diluted 1﹕40 000 and 1﹕8 000, respectively. Under the reaction temperature of 37℃, reaction time of standard substance and monoclonal antibody was 15 min, and 25 min after adding GaMIgG-HRP, also 10 min for termination reaction. Cell line named 2H5 showed a good sensitivity and class-specific toward 10 FQs, the linear regression equation was y= -28.022x+56.219，R²=0.9 782，with an IC₅₀ value of 1.67 ng·mL^-1 for ciprofloxacin, 1.82 ng·mL^-1 for norfloxacin, 1.97 ng·mL^-1 for pefloxacin, 1.54 ng·mL^-1for enrofloxacin, 2.79 ng·mL^-1 for danofloxacin, 3.38 ng·mL^-1 for lomefloxacin, 5.50 ng·mL^-1 for ofloxacin, 4.40 ng·mL^-1 for marbofloxacin, 11.76 ng·mL^-1 for sarafloxacin, 13.60 ng·mL^-1for difloxacin, and the lowest detectable limits (LODs) of 0.09 ng·mL^-1-0.64 ng·mL^-1 and cross- reactivity (CR) of 12.3%-108.4%, no cross-reactivity to other compounds was found. The recovery ranges of 10 FQs spiked in chicken using icELISA were 80.5%-91.8 %, 85.1%-95.7% for HPLC, both of the coefficient variations (CVs) were below 10.0%, and no significant difference (P＞0.05) was observed.【Conclusion】The high-sensitivity and class-specific mAb against FQs was prepared, which laid a solid foundation for FQs multi-residue detection.

Key words: ciprofloxacin, fluoroquinolones, hybridoma cell lines, class-specific monoclonal antibodies, icELISA

李新朋，姜金庆，钱爱东，王自良，范国英，单晓峰，康元环，李艺. 氟喹诺酮类药物多残留酶联免疫检测方法的建立[J]. 中国农业科学, 2014, 47(23): 4726-2735.

LI Xin-peng, JIANG Jin-qing, QIAN Ai-dong, WANG Zi-liang, FAN Guo-ying, SHAN Xiao-feng, KANG Yuan-huan, LI Yi. Development of an ELISA Method for Multi-Residue Detecting of Fluoroquinolones[J]. Scientia Agricultura Sinica, 2014, 47(23): 4726-2735.

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参考文献

[1] Liu Y Z, Zhao G X, Wang P, Liu J, Zhang H C, Wang J P. Production of the broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12 fluoroquinolones in meat. Journal of Environmental Science and Health B, 2013, 48(2): 139-146.

[2] Burkin M A. Enzyme-linked immunosorbent assays of fluoroquin- olones with selective and group specificities. Food and Agricultural Immunology, 2008, 19(2): 131-140.

[3] 李新朋, 姜金庆, 钱爱东, 丁菡, 李艺. 动物性食品中氟喹诺酮类药物多残留检测方法研究进展. 动物医学进展, 2013, 34(9): 99-103.

Li X P, Jiang J Q, Qian A D, Ding H, Li Y. Research progress of multi-residue detection for fluoroquinolones in animal food. Progress in Veterinary Medicine, 2013, 34(9): 99-103. (in Chinese)

[4] Moema D, Nindi M M, Dube S. Development of a dispersive liquid- liquid microextraction method for the determination of fluoro quinolones in chicken liver by high performance liquid chromatography. Analytica Chimica Acta, 2012, 730: 80-86.

[5] 林保银. 鸡肉中11种喹诺酮类药物多残留的高效液相色谱检测. 色谱, 2009, 27(2): 206-210.

Lin B Y. Multi-residue detection of 11 quinolones in chicken muscel by high performance liquid chromatography with fluorescence detection. Chinese Journal of Chromatography, 2009, 27(2): 206-210. (in Chinese)

[6] Sun H W, Zhao W, He P. Effective separation and simultaneous determination of four fluoroquinolones in milk by CE with SPE. Chromatographia, 2008, 68: 425-429.

[7] Bucknall S, Silverlight J, Coldham N, Thorne L, Jackman R. Anti- bodies to the quinolones and fluoroquinolones for the development of generic and specific immunoassays for detection of these residues in animal products. Food Additives and Contaminants, 2003, 20(3): 221-228.

[8] Huet A C, Charlier C, Tttlemier S A, Singh G, Benrejeb S, Delahaut P. Simultaneous determination of (fluoro)quinolone antibiotics in kidney, marine products, eggs, and muscle by enzyme-linked immunosorbent assay (ELISA). Journal of Agricultural and Food Chemistry, 2006, 54(8): 2822-2827.

[9] Tittlemier S A, Gélinas J M, Dufresne G, Haria M, Querry J, Cleroux C, Ménard C, Delahaut P, Singh G, Fischer-Durand N, Godefroy S B. Development of a direct competitive enzyme-linked immunosorbent assay for the detection of fluoroquinolone residues in shrimp. Food Analytical Methods, 2008, 1: 28-35.

[10] Li Y L, Ji B Q, Chen W, Liu L Q, Xu C L, Peng C F, Wang L B. Production of new class-specific polyclonal antibody for determin- ation of fluoroquinolone antibiotics by indirect competitive ELISA. Food and Agricultural Immunology, 2008, 19(4): 251-264.

[11] Shen W, Li Y Z, Xu X, Yuan M, Wang S. Enzyme-linked immunoso- rbent assay and colloidal gold-based immunochromatographic assay for several (fluoro)quinolones in milk. Microchim Acta, 2011, 173: 307-316.

[12] Zhang H T, Jiang J Q, Wang Z L, Chang X Y, Liu X Y, Wang S H, Zhao K, Chen J S. Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin. Journal of Zhejiang University Science B, 2011, 12(11): 884-891.

[13] Duan J H, Yuan Z H. Development of an indirect competitive ELISA for ciprofloxacin residues in food animal edible tissues. Journal of Agricultural and Food Chemistry, 2001, 49: 1087-1089.

[14] 魏东, 刘英. 氟喹诺酮类药物残留多元快速检测试纸条的研制. 核农学报, 2012, 26(9): 1278-1283.

Wei D, Liu Y. Development of a rapid colloidal gold test strip for detecting fluoroquinolones residues. Journal of Nuclear Agricultural Sciences, 2012, 26(9): 1278-1283. (in Chinese)

[15] Pinacho D G, Sa?nchez-Baeza F, Marco M. P. Molecular modeling assisted Hapten design to produce broad selectivity antibodies for fluoroquinolone antibiotics. Analytical Chemistry, 2012, 84(10): 4527-4534.

[16] Hu K, Huang X Y, Jiang Y S, Fang W, Yang X L. Monoclonal antibody based enzymelinked immunosorbent assay for the specific detection of ciprofloxacin and enrofloxacin residues in fishery products. Aquaculture, 2010, 310: 8-12.

[17] Wang Z H, Zhu Y, Ding S Y, He F Y, Beier R C, Li J C, Jiang H Y, Feng C W, Wan Y P, Zhang S X, Kai Z P, Yang X L, Shen J Z. Development of a monoclonal antibody-based broad-specificity ELISA for fluoroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds. Analytical Chemistry, 2007, 79: 4471-4483.

[18] Leivo J, Chappuis C, Lamminmäki U, Lövgren T, Vehniäinen M. En- gineering of a broad-specificity antibody: Detection of eight fluoroq- uinolone antibiotics simultaneously. Analytical Chemistry, 2011, 409: 14-21.

[19] Jiang J Q, Zhang H T, Wang Z L. Multiresidue determination of sar- afloxacin, difloxacin, norfloxacin, and pefloxacin in fish using an enzyme-linked immunosorbent assay. Procedia Environmental Sciences, 2011, 8: 301-306.

[20] 孙远明, 张挺, 雷红涛, 沈玉栋, 杨金易, 王弘. 环丙沙星半抗原、人工抗原和抗体及其制备方法和应用. 中国专利, 20101025693 9. 6[P], 2010- 08-07.

Sun Y M, Zhang T, Lei H T, Shen Y D, Yang J Y. Preparation and application of hapten, artificial antigen, antibody for ciprofloxacin. Chinese Patent, 201010256939. 6[P], 2010- 08-07. (in Chinese)

[21] 姜金庆, 张海棠, 李广领, 王自良, 王建华. 19-去甲睾酮人工抗原及免疫学特性. 农业生物技术学报, 2010, 18(5): 1013-1018.

Jiang J Q, Zhang H T, Li G L, Wang Z L, Wang J H. Preparation of antyficial antigen and immunological trait of 19-nortestosterone. Journal of Agricultural Biotechnology, 2010, 18(5): 1013-1018. (in Chinese)

[22] 职爱民, 李青梅, 刘庆堂, 刘宣兵, 杨继飞, 杨素珍, 柴书军, 杨艳艳, 邓瑞广, 张改平. 抗庆大霉素单克隆抗体的制备及其初步应用. 中国农业科学, 2010, 43(12): 2584-2589.

Zhi A M, Li Q M, Liu Q T, Liu X B, Yang J F, Yang S Z, Chai S J, Yang Y Y, Deng R G, Zhang G P. Preparation and preliminary application of anti-gentamicin monoclonal antibody. Scientia Agricultura Sinica, 2010, 43(12): 2584-2589. (in Chinese)

[23] 王玲玲, 职爱民, 杨艳艳, 宋春美, 王坤, 柴书军, 侯玉泽, 邓瑞广, 张改平. 抗百菌清单克隆抗体的研制与鉴定. 中国农业科学, 2013, 46(7): 1509-1515.

Wang L L, Zhi A M, Yang Y Y, Song C M, Wang K, Chai S J, Hou Y Z,

Deng R G, Zhang G P. Preparation and identification of the monoclonal antibody against chlorothalonil. Scientia Agricultura Sinica, 2013, 46(7): 1509-1515. (in Chinese)

[24] Jiang J Q, Zhang H T, Fan G Y, Ma J Y, Wang Z L, Wang J H. Preparation of monoclonal antibody based indirect competitive ELISA for detecting 19-nortestosterone residue. Chinese Science Bulletin, 2011, 56(25): 2698-2705.

[25] Yuan Z H, Duan J H, Fan S X, Kong K. Comparison of an ELISA and a HPLC for determination of ciprofloxacin residues in pork. Food and Agricultural Immunology, 2001, 13: 199-204.