呋喃西林代谢物单克隆抗体及荧光免疫层析试纸条的制备
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摘要
为建立一种基于荧光免疫层析技术的呋喃西林代谢物的快速检测方法,采用活性酯法合成呋喃西林代谢物(氨基脲盐酸盐)(semicarbazide hydrochloride,SEM)人工抗原,并获得SEM的单克隆抗体,通过将量子点微球(quantumdot submicrobeads,QBs)与SEM偶联,得到QBs探针,制备荧光免疫层析试纸条。结果表明:SEM免疫荧光层析试纸条检测鱼肉组织的检测限为0.247?μg/L,不同添加质量浓度的回收率均在70%～120%之间,批内、批间变异系数均在15%以下,符合我国动物源食品中兽药残留检测方法相关指导文件的规定,且在2～8℃条件下可以保存6个月以上,稳定性良好。
In order to establish a rapid fluorescence immunochromatography method for the detection of semicarbazide hydrochloride(SEM) as a furacilin metabolite, SEM hapten was synthesized and conjugated to bovine serum albumin(BSA) and ovalbumin(OVA) by the active ester method, and the hapten-protein conjugates were employed to obtain antiSEM monoclonal antibody. Meanwhile, SEM was conjugated to quantumdot microspheres, yielding a probe. Finally,a?fluorescence?immunochromatography?test?strip?was?prepared.?The?results?showed?that?the?detection?limit?of?the?test?strip?for?fish?meat?was?0.247?μg/L.?The?recoveries?of?SEM?from?blank?fish?samples?spiked?at?different?concentrations?were?between?70% and 120%,?and?within?and?between?batches?coefficients?of?variation(CV)?were?below?15%, which was in accordance with the requirements for the determination of veterinary drug residues in animal-derived foods. The test strip was stable at2–8 ℃ for up to 6 months.
In order to establish a rapid fluorescence immunochromatography method for the detection of semicarbazide hydrochloride(SEM) as a furacilin metabolite, SEM hapten was synthesized and conjugated to bovine serum albumin(BSA) and ovalbumin(OVA) by the active ester method, and the hapten-protein conjugates were employed to obtain antiSEM monoclonal antibody. Meanwhile, SEM was conjugated to quantumdot microspheres, yielding a probe. Finally,a?fluorescence?immunochromatography?test?strip?was?prepared.?The?results?showed?that?the?detection?limit?of?the?test?strip?for?fish?meat?was?0.247?μg/L.?The?recoveries?of?SEM?from?blank?fish?samples?spiked?at?different?concentrations?were?between?70% and 120%,?and?within?and?between?batches?coefficients?of?variation(CV)?were?below?15%, which was in accordance with the requirements for the determination of veterinary drug residues in animal-derived foods. The test strip was stable at2–8 ℃ for up to 6 months.
引文
[1]李军,张会彩,刘聚祥,等.酶联免疫吸附检测动物饲料中呋喃唑酮[J].中国兽医杂志,2009,45(8):85-86.DOI:10.3969/j.issn.0529-6005.2009.08.034.
[2]蒋宏伟.酶联免疫技术在动物产品中硝基呋喃类药物残留检测的应用[J].陕西农业科学,2006(5):53-55.
[3]祝伟霞,刘亚风,梁伟,等.动物性食品中硝基呋喃类药物残留检测研究进展[J].动物医学进展,2010,31(2):99-102.DOI:10.3969/j.issn.1007-5038.2010.02.023.
[4]中华人民共和国农业部第235号公告[EB/OL].(2002-12-24)[2018-12-10].
[5]LAI W H,FUNG D Y,XU Y,et al.Screening procedures for clenbuterol?residue?determination?in?law?swine?livers?using?lateral-flow?assay and enzyme-linked immunosorbent assay[J].Journal of Food Protection,2008,71(4):865-869.
[6]于慧娟,蔡友琼,毕士川,等.高效液相色谱法测定水产品中呋喃唑酮的残留量[J].色谱,2005,23(1):114-117.DOI:10.3321/j.issn:1000-8713.2005.01.031.
[7]彭涛,邱月明,李淑娟,等.高效液相色谱-串联质谱法测定动物肌肉中硝基呋喃类抗生素代谢物[J].检验检疫科学,2003,13(6):23-28.DOI:10.3969/j.issn.1674-5354.2003.06.007.
[8]COOPER K M,CADDELL A.Production and characterization of polyclonal antibodies to a derivative of 3-amino-2-oxazolidinone,a metabolite of the nitrofuran furazolidone[J].Analytica Chimica Acta,2004,520:79-86.DOI:10.1016/j.aca.2004.05.074.
[9]王丽娜,刘凯,杜柏林,等.UPLC/MS/MS测定动物尿液中9种β-受体激动剂残留量[J].现代畜牧兽医,2013(8):42-46.DOI:10.3969/j.issn.1672-9692.2013.08.018.
[10]葛怀礼,王莲,李金卿,等.盐酸克伦特罗荧光定量检测试剂盒质量评价[J].中国畜牧兽医,2014,41(10):285-289.
[11]赵亦静,傅小伟,张明洲,等.呋喃西林免疫抗原的合成与鉴定[J].现代农业科技,2008,14:197-199.DOI:10.3969/j.issn.1007-5739.2008.14.151.
[12]杨立国,胡少昶,魏平华,等.酶免疫测定技术[M].南京:南京大学出版社,1998:189-284.
[13]李春生,刘静静,杜顺丰,等.呋喃唑酮代谢物单克隆抗体和胶体金免疫层析试纸条的研制[J].现代食品科技,2017,43(3):326-331.DOI:10.13982/j.mfst.1673-9078.2017.6.048.
[14]丁桥棋,李丽,范文韬,等.基于新型量子点荧光微球的氯霉素免疫层析试纸条的制备和应用[J].分析化学研究报告,2017,45(11):1686-1693.DOI:10.11895/j.issn.0253-3820.170300.
[15]解泉源,赖卫华,刘春梅,等.大肠杆菌O157:H7荧光微球免疫层析试纸条的研制[J].食品科学,2013,34(16):353-357.DOI:10.7506/spkx1002-6630-201316072.
[16]李军,刘莲娜,孟晓璐,等.硝基呋喃类药物胶体金免疫层析试纸条的研制[J].畜牧与兽医,2014,46(1):74-77.
[17]段宏,陈雪岚,江湖,等.量子点荧光微球免疫层析试纸条定量检测恶性恶性疟原虫[J].分析化学,2015,43(3):338-343.DOI:10.11895i ssn.0253-3820.140848.
[18]ZHANG Mingxia,LI Cun,WU Yinliang.Determination of phenylethanolamine A in animal hair,tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS[J].Journal of Chromatogrephy B,2012,900:94-99.DOI:10.1016/j.jchromb.2012.05.030.
[19]唐海波,齐维,唐诗幸,等.荧光定量快速检测鲜奶中四环素总量残留[J].现代食品科技,2012,28(11):1600-1602.
[20]胡华军,付涛,张明洲,等.CdTe/ZnSe壳核量子点免疫层析试纸条检测科伦特伦的研究[J].分析化学,2010,38(12):1727-1731.DOI:10.3724/SP.J.1096.2010.01727.
[21]郭诗静,唐海波,齐维,等.免疫荧光层析快速检测猪尿中克伦特罗含量[J].现代食品科技,2013,29(5):1154-1156.
[22]方琦,黄锡荣,李凯,等.降钙素原荧光免疫层析定量检测方法的建立及性能评估[J].中华检验医学杂志,2012,35(12):1102-1107.DOI:10.3760/cma.j.issn.1009-9158.2012.12.011.
[23]郭艳宏,李飞,邹明强,等.用于检测氯霉素类残留物的荧光免疫检测试纸条的研制[J].化学试剂,2010,32(6):496-498.DOI:10.3969/j.issn.0258-3283.2010.06.005.
[24]范放,朱海,洪小柳,等.氯霉素荧光纳米颗粒免疫层析法的建立[J].现代农业科学,2009,16(9):13-14.
[25]KE Yan,FU Lanli,HONG Xiafei,et al.Acute clenbuterol induces hypotension,atrioventricular block and cardiacasystole in the rabbit[J].Cardiovasc Toxicol,2013,13(1):85-90.DOI:10.1007/sl2012-012-9185-8.
[26]胡佳丽,于东升,刘小雷,等.荧光免疫层析法快速测定牛奶中头孢氨苄残留量[J].中国食品卫生杂志,2014,26(4):362-366.DOI:10.13590/j.cjfh.2014.04.015.
[27]姜艳彬,孙冠如,王海,等.盐酸克伦特罗胶体金快速检测试纸条的研制[J].中国畜牧兽医,2010,37(10):109-113.
[28]朱海,范放,洪小柳,等.氯霉素荧光纳米颗粒免疫层析法的建立[J].现代农业科学,2009,16(9):13-14.
[29]李丹妮,黄华,张倩,等.3种β-受体激动剂荧光免疫层析试纸条的制备及特性研究[J].中国畜牧兽医,2017,44(8):2437-2442.DOI:10.16431/j.cnki.1671-7236.2017.08.031.
[30]唐勇,王五洲,向军俭,等.呋喃西林代谢物氨基脲免疫吸附生物条码检测方法的初步研究[J].食品科学,2009,30(18):274-277.
[31]何方洋,冯才伟,孙震,等.饲料中硝基呋喃类药物免疫胶体金检测试纸条的研制[J].饲料工业,2012,33(23):35-38.
[32]赵正苗,罗晓琴,汪善良,等.应用胶体金免疫层析法检测动物组织中呋喃西林代谢物的残留[J].上海畜牧兽医通讯,2012(5):4-5.
[2]蒋宏伟.酶联免疫技术在动物产品中硝基呋喃类药物残留检测的应用[J].陕西农业科学,2006(5):53-55.
[3]祝伟霞,刘亚风,梁伟,等.动物性食品中硝基呋喃类药物残留检测研究进展[J].动物医学进展,2010,31(2):99-102.DOI:10.3969/j.issn.1007-5038.2010.02.023.
[4]中华人民共和国农业部第235号公告[EB/OL].(2002-12-24)[2018-12-10].
[5]LAI W H,FUNG D Y,XU Y,et al.Screening procedures for clenbuterol?residue?determination?in?law?swine?livers?using?lateral-flow?assay and enzyme-linked immunosorbent assay[J].Journal of Food Protection,2008,71(4):865-869.
[6]于慧娟,蔡友琼,毕士川,等.高效液相色谱法测定水产品中呋喃唑酮的残留量[J].色谱,2005,23(1):114-117.DOI:10.3321/j.issn:1000-8713.2005.01.031.
[7]彭涛,邱月明,李淑娟,等.高效液相色谱-串联质谱法测定动物肌肉中硝基呋喃类抗生素代谢物[J].检验检疫科学,2003,13(6):23-28.DOI:10.3969/j.issn.1674-5354.2003.06.007.
[8]COOPER K M,CADDELL A.Production and characterization of polyclonal antibodies to a derivative of 3-amino-2-oxazolidinone,a metabolite of the nitrofuran furazolidone[J].Analytica Chimica Acta,2004,520:79-86.DOI:10.1016/j.aca.2004.05.074.
[9]王丽娜,刘凯,杜柏林,等.UPLC/MS/MS测定动物尿液中9种β-受体激动剂残留量[J].现代畜牧兽医,2013(8):42-46.DOI:10.3969/j.issn.1672-9692.2013.08.018.
[10]葛怀礼,王莲,李金卿,等.盐酸克伦特罗荧光定量检测试剂盒质量评价[J].中国畜牧兽医,2014,41(10):285-289.
[11]赵亦静,傅小伟,张明洲,等.呋喃西林免疫抗原的合成与鉴定[J].现代农业科技,2008,14:197-199.DOI:10.3969/j.issn.1007-5739.2008.14.151.
[12]杨立国,胡少昶,魏平华,等.酶免疫测定技术[M].南京:南京大学出版社,1998:189-284.
[13]李春生,刘静静,杜顺丰,等.呋喃唑酮代谢物单克隆抗体和胶体金免疫层析试纸条的研制[J].现代食品科技,2017,43(3):326-331.DOI:10.13982/j.mfst.1673-9078.2017.6.048.
[14]丁桥棋,李丽,范文韬,等.基于新型量子点荧光微球的氯霉素免疫层析试纸条的制备和应用[J].分析化学研究报告,2017,45(11):1686-1693.DOI:10.11895/j.issn.0253-3820.170300.
[15]解泉源,赖卫华,刘春梅,等.大肠杆菌O157:H7荧光微球免疫层析试纸条的研制[J].食品科学,2013,34(16):353-357.DOI:10.7506/spkx1002-6630-201316072.
[16]李军,刘莲娜,孟晓璐,等.硝基呋喃类药物胶体金免疫层析试纸条的研制[J].畜牧与兽医,2014,46(1):74-77.
[17]段宏,陈雪岚,江湖,等.量子点荧光微球免疫层析试纸条定量检测恶性恶性疟原虫[J].分析化学,2015,43(3):338-343.DOI:10.11895i ssn.0253-3820.140848.
[18]ZHANG Mingxia,LI Cun,WU Yinliang.Determination of phenylethanolamine A in animal hair,tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS[J].Journal of Chromatogrephy B,2012,900:94-99.DOI:10.1016/j.jchromb.2012.05.030.
[19]唐海波,齐维,唐诗幸,等.荧光定量快速检测鲜奶中四环素总量残留[J].现代食品科技,2012,28(11):1600-1602.
[20]胡华军,付涛,张明洲,等.CdTe/ZnSe壳核量子点免疫层析试纸条检测科伦特伦的研究[J].分析化学,2010,38(12):1727-1731.DOI:10.3724/SP.J.1096.2010.01727.
[21]郭诗静,唐海波,齐维,等.免疫荧光层析快速检测猪尿中克伦特罗含量[J].现代食品科技,2013,29(5):1154-1156.
[22]方琦,黄锡荣,李凯,等.降钙素原荧光免疫层析定量检测方法的建立及性能评估[J].中华检验医学杂志,2012,35(12):1102-1107.DOI:10.3760/cma.j.issn.1009-9158.2012.12.011.
[23]郭艳宏,李飞,邹明强,等.用于检测氯霉素类残留物的荧光免疫检测试纸条的研制[J].化学试剂,2010,32(6):496-498.DOI:10.3969/j.issn.0258-3283.2010.06.005.
[24]范放,朱海,洪小柳,等.氯霉素荧光纳米颗粒免疫层析法的建立[J].现代农业科学,2009,16(9):13-14.
[25]KE Yan,FU Lanli,HONG Xiafei,et al.Acute clenbuterol induces hypotension,atrioventricular block and cardiacasystole in the rabbit[J].Cardiovasc Toxicol,2013,13(1):85-90.DOI:10.1007/sl2012-012-9185-8.
[26]胡佳丽,于东升,刘小雷,等.荧光免疫层析法快速测定牛奶中头孢氨苄残留量[J].中国食品卫生杂志,2014,26(4):362-366.DOI:10.13590/j.cjfh.2014.04.015.
[27]姜艳彬,孙冠如,王海,等.盐酸克伦特罗胶体金快速检测试纸条的研制[J].中国畜牧兽医,2010,37(10):109-113.
[28]朱海,范放,洪小柳,等.氯霉素荧光纳米颗粒免疫层析法的建立[J].现代农业科学,2009,16(9):13-14.
[29]李丹妮,黄华,张倩,等.3种β-受体激动剂荧光免疫层析试纸条的制备及特性研究[J].中国畜牧兽医,2017,44(8):2437-2442.DOI:10.16431/j.cnki.1671-7236.2017.08.031.
[30]唐勇,王五洲,向军俭,等.呋喃西林代谢物氨基脲免疫吸附生物条码检测方法的初步研究[J].食品科学,2009,30(18):274-277.
[31]何方洋,冯才伟,孙震,等.饲料中硝基呋喃类药物免疫胶体金检测试纸条的研制[J].饲料工业,2012,33(23):35-38.
[32]赵正苗,罗晓琴,汪善良,等.应用胶体金免疫层析法检测动物组织中呋喃西林代谢物的残留[J].上海畜牧兽医通讯,2012(5):4-5.