水产品中恩诺沙星胶体金免疫层析毛细管检测及其前处理
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摘要
目的:结合胶体金标记技术和免疫层析技术建立胶体金免疫层析毛细管检测技术,用于水产品中恩诺沙星残留的快速现场检测。方法:以间接竞争的实验原理,通过优化检测区抗原、质控区二抗、金标一抗检测浓度初步建立胶体金免疫层析毛细管检测方法,并进一步确定其检测灵敏度、特异性、重复性等及前处理的优化操作。结果:该方法检测恩诺沙星的视觉检测限为5 ng/mL,半定量检测限为1.29 ng/mL。在大菱鲆、鲅鱼、舌鳎、南美白对虾阴性样品中的平均添加回收率在78.3%~129%之间,相对标准偏差均小于10%。特异性实验表明,除对环丙沙星外,对其他相似物的特异性较好;环丙沙星的视觉检测限为10 ng/mL,半定量检测限为4.37 ng/mL,满足国标检测的限量要求。同时,针对免疫层析毛细管检测的前处理方法进行初步探究,最终确定采用常温条件下磺基水杨酸提取的前处理方法。结论:恩诺沙星胶体金免疫层析毛细管检测技术具有简单、快速、易操作、重复性好的优势,为食品药物残留检测领域提供了检测新方法。同时,选择新型试剂作为胶体金免疫层析毛细管检测技术的前处理方法,该方法可以为多种快检技术的前处理提供理论基础与技术支撑。
Objective: To establish a nano-gold capillary immunochromatographic assay(CICA) based on a combination of colloidal-gold labeling and Immunochromatography for the rapid field detection of enrofloxacin(EF) residues in aquatic products. Methods: Based on the principle of indirect competitive immunoreaction, the CICA method was developed by optimizing antigen concentration in the test area, secondary antibody in the control area and gold-labeled antibody standard concentration. Then the sensitivity, specificity and repeatability of this method and several sample pretreatments were evaluated. Results: The visual and semi-quantitative detection limits for EF were estimated to be 5 and 1.29 ng/mL, respectively. The recoveries of EF in spiked negative samples of turbot, Spanish mackerel, tonguefishes, and Pacific while shrimp(P. vannamei) were 78.3%–129% and The intra-and inter-assay precision expressed as relative standard deviation(RSD) was less than 10%. Specificity analysis indicated that this method had strong specificity to all analogues except ciprofloxacin(CIP), for which the visual and semi-quantitative detection limit were 10 and 4.37 ng/mL, respectively and met the requirement of the national standard of China. Sulfonic acid extraction under normal temperature conditions was chosen as the optimal pretreatment method for CICA. Conclusion: CICA was a simple, rapid, easy-to-operate and reproducible method and could provide a new approach for the detection of antibiotic residues in aquatic products. Meanwhile, the pretreatment method for CICA chosen in this study can provide a theoretical basis and technical support for other rapid detection methods.
Objective: To establish a nano-gold capillary immunochromatographic assay(CICA) based on a combination of colloidal-gold labeling and Immunochromatography for the rapid field detection of enrofloxacin(EF) residues in aquatic products. Methods: Based on the principle of indirect competitive immunoreaction, the CICA method was developed by optimizing antigen concentration in the test area, secondary antibody in the control area and gold-labeled antibody standard concentration. Then the sensitivity, specificity and repeatability of this method and several sample pretreatments were evaluated. Results: The visual and semi-quantitative detection limits for EF were estimated to be 5 and 1.29 ng/mL, respectively. The recoveries of EF in spiked negative samples of turbot, Spanish mackerel, tonguefishes, and Pacific while shrimp(P. vannamei) were 78.3%–129% and The intra-and inter-assay precision expressed as relative standard deviation(RSD) was less than 10%. Specificity analysis indicated that this method had strong specificity to all analogues except ciprofloxacin(CIP), for which the visual and semi-quantitative detection limit were 10 and 4.37 ng/mL, respectively and met the requirement of the national standard of China. Sulfonic acid extraction under normal temperature conditions was chosen as the optimal pretreatment method for CICA. Conclusion: CICA was a simple, rapid, easy-to-operate and reproducible method and could provide a new approach for the detection of antibiotic residues in aquatic products. Meanwhile, the pretreatment method for CICA chosen in this study can provide a theoretical basis and technical support for other rapid detection methods.
引文
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[7]李怀明.盐酸克伦特罗及恩诺沙星荧光微球免疫层析试纸条定量检测方法的建立[D].南昌:南昌大学,2012:32-41.
[8]龚云飞,邹晓楠,张露露,等.化学发光免疫法检测食品中恩诺沙星残留的研究[J].食品工业科技,2014,35(4):66-69.DOI:10.13386/j.issn1002-0306.2014.04.046.
[9]QU X L,LIN H,DU S Y,et al.Development of a nano-gold capillary immunochromatographic assay for rapid and semi-quantitative detection of clenbuterol residues[J].Food Analytical Methods,2016,9(9):1-10.DOI:10.1007/s12161-016-0442-5.
[10]杜淑媛.水产品中危害物胶体金免疫层析毛细管检测技术的研究[D].青岛:中国海洋大学,2015:25-90.
[11]李德亮,王军,常志显,等.二氧化硅表面的GPTMS修饰[J].化学进展,2008,20(7):1115-1121.
[12]GIOVANNOLI C,ANFOSSIA L,BAGGIANI C,et al.A novel approach for a non-competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin[J].Journal of Chromatography A,2007,1155(2):187-192.DOI:10.1016/j.chroma.2007.02.056.
[13]杨玉新,叶阳,周有祥,等.四种化学还原法制备胶体金的比较研究[J].湖北农业科学,2011,50(3):476-478.DOI:10.3969/j.issn.0439-8114.2011.03.013.
[14]SLOT J W,GEUZE H J.A new method of preparing gold probes for multiple-labeling cytochemistry[J].European Journal of Cell Biology,1985,38(1):87-93.
[15]HONG D D,SOHN O J,LAM H T,et al.An optical p H sensor with extended detection range based on fluoresceinamine covalently bound to sol-gel support[J].Microchemical Journal,2006,84(1/2):50-55.DOI:10.1016/j.microc.2006.04.013.
[16]崔梦琪.基于磺基水杨酸的水产品氟喹诺酮类药物酶联免疫检测前处理技术研究[D].青岛:中国海洋大学,2015:30-43.
[17]陆宗超.大菱鲆过敏原小清蛋白的制备及氧化对其Ig E结合能力的影响[D].青岛:中国海洋大学,2013:15-17.
[18]刘彦君,谭婧,章崇杰,等.应用曲线拟合方法求解单克隆抗体亲和常数中OD_(100)[J].西部医学,2010,22(4):599-602.
[19]BEATTY J D,BEATTY B G,VLAHOS W G.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].Journal of Immunological Methods,1987,100(1/2):173-179.DOI:10.1016/0022-1759(87)90187-6.
[20]马爱团,钟秀会,史万玉,等.胶体金标记抗体的制备及DotIGSS检测IBDV方法的建立[J].黑龙江畜牧兽医,2008(2):65-67.DOI:10.3969/j.issn.1004-7034.2008.02.027.
[21]宋宏新,邹联柱,李韵,等.纳米胶体金的制备及其抗体标记研究[J].食品科技,2011,36(3):249-252.DOI:10.13684/j.cnki.spkj.2011.03.001.
[22]ZHAO Y L,ZHANG G P,LIU Q T,et al.Development of a lateral flow colloidal gold immunoassay strip for the rapid detection of enrofloxacin residues[J].Journal of Agricultural&Food Chemistry,2008,56(24):12138-12142.DOI:10.1021/jf802648z.
[23]刘珊娜,葛怀娜,孟繁桐,等.李斯特菌免疫检测用胶体金的制备及抗体标记[J].食品科技,2016,41(1):321-324.DOI:10.13684/j.cnki.spkj.2016.01.063.
[24]BLAZKOVA M,MICKOVAHOLUBOVA B,RAUCH P,et al.Immunochromatographic colloidal carbon-based assay for detection of methiocarb in surface water[J].Biosensors&Bioelectronics,2009,25(4):753-758.DOI:10.1016/j.bios.2009.08.023.
[25]吕涛,冯奇,史利涛,等.分析方法检出限的确定[J].漯河职业技术学院学报,2007,6(4):199-200.
[26]姜金庆,杨雪峰,王自良,等.氟喹诺酮类药物多残留间接竞争ELISA检测方法的建立[J].中国预防兽医学报,2011,33(11):887-892.
[27]王晓艳,林纪昀,杨利,等.两株高特异性抗恩诺沙星单克隆抗体的研制[J].食品安全质量检测学报,2010,27(1):5-10.
[28]农业部兽医局.动物性食品中兽药最高残留限量:中华人民共和国农业部公告第235号[EB/OL].(2002-12-24).http://www.moa.gov.cn/zwllm/tzgg/gg/200302/t20030226_59300.htm.
[29]王秀丹.鱼类主要基质组分对药物残留酶联免疫吸附检测的影响[D].青岛:中国海洋大学,2014:14-31.
[30]李桂芝,高福凯,张兴梅,等.柠檬酸水溶液生物亲和萃取-高效液相色谱法检测鱼肉样品中喹诺酮类药物残留[J].分析化学,2013,41(10):1592-1596.DOI:10.3724/SP.J.1096.2013.30385.
[31]徐锐.海产品中恩诺沙星残留的免疫胶体金层析现场快速检测技术[D].青岛:中国海洋大学,2013:12-17.
[32]PETRUCCELLI S,ANON M C.Thermal aggregation of soy protein isolates[J].Journal of Agricultural and Food Chemistry,1995,43(12):3035-3041.DOI:10.1021/jf00060a009.
[33]郭凤仙.热处理对大豆分离蛋白结构及功能特性的影响[D].无锡:江南大学,2009:8-28.
[34]梅彬.水产品中恩诺沙星胶体金快速检测试纸条的研制[D].厦门:集美大学,2015:12-41.
[35]徐锐.海产品中恩诺沙星残留的免疫胶体金层析现场快速检测技术[D].青岛:中国海洋大学,2013:28-37.
[36]王宵雪.恩诺沙星免疫胶体金快速检测试纸条的研制[D].天津:天津科技大学,2012:34-45.
[37]樊晓博,谢兰心.酶标抗原直接竞争ELISA检测食品中氟喹诺酮类药物多残留[J].食品科学,2015,36(24):265-269.DOI:10.7506/spkx1002-6630-201524049.
[2]叶建美,吴康,吴洪丽,等.恩诺沙星抗菌效果及药代动力学研究进展[J].湖北农业科学,2015,54(23):5813-5816.DOI:10.14088/j.cnki.issn0439-8114.2015.23.005.
[3]CINQUINA A L,ROBERTI P,GIANNETTI L,et al.Determination of enrofloxacin and its metabolite ciprofloxacin in goat milk by highperformance liquid chromatography with diode-array detection:optimization and validation[J].Journal of Chromatography A,2003,987(1):221-226.DOI:10.1016/S0021-9673(02)01800-9.
[4]褚洪蕊,唐景春.农产品中农兽药残留检测技术与应用研究[J].农业环境科学学报,2007,26(B10):656-661.
[5]张冬冬,苏超,洪伟,等.动物源食品中氟喹诺酮类兽药残留检测的研究进展[J].安徽农学通报,2016,22(13):38-41.DOI:10.3969/j.issn.1007-7731.2016.13.014.
[6]潘明飞,王俊平,方国臻,等.食品中农兽药残留检测新技术研究进展[J].食品科学,2014,35(15):277-282.DOI:10.7506/spkx1002-6630-201415056.
[7]李怀明.盐酸克伦特罗及恩诺沙星荧光微球免疫层析试纸条定量检测方法的建立[D].南昌:南昌大学,2012:32-41.
[8]龚云飞,邹晓楠,张露露,等.化学发光免疫法检测食品中恩诺沙星残留的研究[J].食品工业科技,2014,35(4):66-69.DOI:10.13386/j.issn1002-0306.2014.04.046.
[9]QU X L,LIN H,DU S Y,et al.Development of a nano-gold capillary immunochromatographic assay for rapid and semi-quantitative detection of clenbuterol residues[J].Food Analytical Methods,2016,9(9):1-10.DOI:10.1007/s12161-016-0442-5.
[10]杜淑媛.水产品中危害物胶体金免疫层析毛细管检测技术的研究[D].青岛:中国海洋大学,2015:25-90.
[11]李德亮,王军,常志显,等.二氧化硅表面的GPTMS修饰[J].化学进展,2008,20(7):1115-1121.
[12]GIOVANNOLI C,ANFOSSIA L,BAGGIANI C,et al.A novel approach for a non-competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin[J].Journal of Chromatography A,2007,1155(2):187-192.DOI:10.1016/j.chroma.2007.02.056.
[13]杨玉新,叶阳,周有祥,等.四种化学还原法制备胶体金的比较研究[J].湖北农业科学,2011,50(3):476-478.DOI:10.3969/j.issn.0439-8114.2011.03.013.
[14]SLOT J W,GEUZE H J.A new method of preparing gold probes for multiple-labeling cytochemistry[J].European Journal of Cell Biology,1985,38(1):87-93.
[15]HONG D D,SOHN O J,LAM H T,et al.An optical p H sensor with extended detection range based on fluoresceinamine covalently bound to sol-gel support[J].Microchemical Journal,2006,84(1/2):50-55.DOI:10.1016/j.microc.2006.04.013.
[16]崔梦琪.基于磺基水杨酸的水产品氟喹诺酮类药物酶联免疫检测前处理技术研究[D].青岛:中国海洋大学,2015:30-43.
[17]陆宗超.大菱鲆过敏原小清蛋白的制备及氧化对其Ig E结合能力的影响[D].青岛:中国海洋大学,2013:15-17.
[18]刘彦君,谭婧,章崇杰,等.应用曲线拟合方法求解单克隆抗体亲和常数中OD_(100)[J].西部医学,2010,22(4):599-602.
[19]BEATTY J D,BEATTY B G,VLAHOS W G.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].Journal of Immunological Methods,1987,100(1/2):173-179.DOI:10.1016/0022-1759(87)90187-6.
[20]马爱团,钟秀会,史万玉,等.胶体金标记抗体的制备及DotIGSS检测IBDV方法的建立[J].黑龙江畜牧兽医,2008(2):65-67.DOI:10.3969/j.issn.1004-7034.2008.02.027.
[21]宋宏新,邹联柱,李韵,等.纳米胶体金的制备及其抗体标记研究[J].食品科技,2011,36(3):249-252.DOI:10.13684/j.cnki.spkj.2011.03.001.
[22]ZHAO Y L,ZHANG G P,LIU Q T,et al.Development of a lateral flow colloidal gold immunoassay strip for the rapid detection of enrofloxacin residues[J].Journal of Agricultural&Food Chemistry,2008,56(24):12138-12142.DOI:10.1021/jf802648z.
[23]刘珊娜,葛怀娜,孟繁桐,等.李斯特菌免疫检测用胶体金的制备及抗体标记[J].食品科技,2016,41(1):321-324.DOI:10.13684/j.cnki.spkj.2016.01.063.
[24]BLAZKOVA M,MICKOVAHOLUBOVA B,RAUCH P,et al.Immunochromatographic colloidal carbon-based assay for detection of methiocarb in surface water[J].Biosensors&Bioelectronics,2009,25(4):753-758.DOI:10.1016/j.bios.2009.08.023.
[25]吕涛,冯奇,史利涛,等.分析方法检出限的确定[J].漯河职业技术学院学报,2007,6(4):199-200.
[26]姜金庆,杨雪峰,王自良,等.氟喹诺酮类药物多残留间接竞争ELISA检测方法的建立[J].中国预防兽医学报,2011,33(11):887-892.
[27]王晓艳,林纪昀,杨利,等.两株高特异性抗恩诺沙星单克隆抗体的研制[J].食品安全质量检测学报,2010,27(1):5-10.
[28]农业部兽医局.动物性食品中兽药最高残留限量:中华人民共和国农业部公告第235号[EB/OL].(2002-12-24).http://www.moa.gov.cn/zwllm/tzgg/gg/200302/t20030226_59300.htm.
[29]王秀丹.鱼类主要基质组分对药物残留酶联免疫吸附检测的影响[D].青岛:中国海洋大学,2014:14-31.
[30]李桂芝,高福凯,张兴梅,等.柠檬酸水溶液生物亲和萃取-高效液相色谱法检测鱼肉样品中喹诺酮类药物残留[J].分析化学,2013,41(10):1592-1596.DOI:10.3724/SP.J.1096.2013.30385.
[31]徐锐.海产品中恩诺沙星残留的免疫胶体金层析现场快速检测技术[D].青岛:中国海洋大学,2013:12-17.
[32]PETRUCCELLI S,ANON M C.Thermal aggregation of soy protein isolates[J].Journal of Agricultural and Food Chemistry,1995,43(12):3035-3041.DOI:10.1021/jf00060a009.
[33]郭凤仙.热处理对大豆分离蛋白结构及功能特性的影响[D].无锡:江南大学,2009:8-28.
[34]梅彬.水产品中恩诺沙星胶体金快速检测试纸条的研制[D].厦门:集美大学,2015:12-41.
[35]徐锐.海产品中恩诺沙星残留的免疫胶体金层析现场快速检测技术[D].青岛:中国海洋大学,2013:28-37.
[36]王宵雪.恩诺沙星免疫胶体金快速检测试纸条的研制[D].天津:天津科技大学,2012:34-45.
[37]樊晓博,谢兰心.酶标抗原直接竞争ELISA检测食品中氟喹诺酮类药物多残留[J].食品科学,2015,36(24):265-269.DOI:10.7506/spkx1002-6630-201524049.