直接竞争ELISA法检测动物源食品中利巴韦林残留
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摘要
本研究针对动物源食品中利巴韦林残留的问题,建立了直接竞争酶联免疫分析方法,快速高效地检测利巴韦林残留。采用活泼酯法将利巴韦林(RBV)与钥孔血蓝蛋白(KLH)偶联制备利巴韦林完全抗原(KLH-RBV),通过免疫新西兰大耳白兔得到多抗血清,经Protein A-Sepharose 4B凝胶柱纯化后得到纯化抗体,在此基础上建立直接竞争ELISA检测方法。该方法的IC_(50)为10.84 ng/mL,最低检测限达0.002 ng/mL,对鸡肉、鸡肝、猪肉、猪肝等实际样品进行检测,加标回收率为80.23%～90.07%。采用高效液相色谱法进行验证,两种方法测定结果呈现良好的线性关系(R~2≥0.99)。本文建立的方法灵敏度高,简便快速,适合现场大批量快速检测动物源食品中利巴韦林残留。
Aiming at the problem of ribavirin residues in animal source foods,a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was established to detect ribavirin residues rapidly and efficiently. Ribavirin (RBV) was conjugated with keyhole limpet hemoeyanin (KLH) by active ester method to prepare the ribavirin complete antigen (KLH-RBV). The antiserum was purified with Protein A-Sepharose 4 B to prepare polyclonal antibody against ribavirin. A dc-ELISA was developed to detect and quantitate ribavirin with IC_(50) of 10.84 ng/mL and detection limit of 0.002 ng/mL. This method was applied to determine RBV in real chicken,chicken liver,pork and pig liver samples with satisfactory result of recovery rate ranged from 80.23% to 90.07%. HPLC was used to verify the results and the results of the two methods showed a good linear relationship. The method established was sensitive and the procedure of sample pretreatment was simple and quick. It was suitable for spot rapid detection of RBV residue in animal source foods.
Aiming at the problem of ribavirin residues in animal source foods,a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was established to detect ribavirin residues rapidly and efficiently. Ribavirin (RBV) was conjugated with keyhole limpet hemoeyanin (KLH) by active ester method to prepare the ribavirin complete antigen (KLH-RBV). The antiserum was purified with Protein A-Sepharose 4 B to prepare polyclonal antibody against ribavirin. A dc-ELISA was developed to detect and quantitate ribavirin with IC_(50) of 10.84 ng/mL and detection limit of 0.002 ng/mL. This method was applied to determine RBV in real chicken,chicken liver,pork and pig liver samples with satisfactory result of recovery rate ranged from 80.23% to 90.07%. HPLC was used to verify the results and the results of the two methods showed a good linear relationship. The method established was sensitive and the procedure of sample pretreatment was simple and quick. It was suitable for spot rapid detection of RBV residue in animal source foods.
引文
[1]程忠刚,傅伟龙,刘树中.动物抗病毒药物的应用研究进展[J].兽药导刊,2010(8):42-45.
[2]谭胜国.因误用利巴韦林治理猪流感而引发药物中毒的诊疗报告[J].当代畜牧,2014(30):31-35.
[3]中华人民共和国农业部.中华人民共和国农业部公告第560号[Z].农业部公告[2005]560号.北京:农业部,2005-10-28.
[4]周剑,王敏,汤晓艳,等.畜禽产品监测用抗病毒类药物标准物质研制与应用情况[J].化学分析计量,2013,22(4):1-3.
[5]RK Austin,PE Trefts,M Hintz,et al.Sensitive radioimmunoassay for the broad-spectrum antiviral agent ribavirin[J].Antimicrobial Agents & Chemotherapy,1983,24(5):696-701.
[6]Melendez M,Rosario O,Zayas B,et al.HPLC-MS/MS method for the intracellular determination of ribavirin momophosphate and ribavirin triphosphate in CEMss cells[J].J Pharm Biomed Anal,2009,49(5):1233-240.
[7]D’Avolio A,Iba?nez A,Sciandra M,et al.Validation of liquid/liquid extraction method coupled with HPLC-UV for measurement of ribavirin plasma levels in HCV-positive patients[J].Journal of Chromatography B,2006,835(1):127-130.
[8]Homma M,Jayewardene A L,Gambertoglio J,et al.High-performance liquid chromatographic determination of ribavirin in whole blood to assess disposition in erythrocyte[J].Antimicrobial Agents and Chemotherapy,1999,43(11):2716-2719.
[9]Page T,Conor J D.The metabolism of ribavirin in erythrocytes and nucleated cells[J].International Journal of Biochemistry,1990,22(4):379-383.
[10]Berendsen B J A,Wegh R S,Essers M L,et al.Quantitative trace analysis of a broad range of antiviral drugs in poultry muscle using column-switch liquid chromatography coupled to tandem mass spectrometry[J].Analytical and Bioanalytical Chemistry,2012(4):1611-623.
[11]Zhang J Z,Zhao J,Zhou J H,et al.Determination of amantadine residue in honey by solid-phase extraction and high-performance liquid chromatography with pre-olumnderivatization and fluorometric detection[J].Chinese Journal of Chemistry,2011(8):1764-768.
[12]Michael C B,Regula T,Wolfgang T.Determination of ribavirin in human serum and plasma by capillary electrophoresis[J].Electrophoresis,2004,25(6):7-8.
[13]汪永忠,张国栋,吕布.高效毛细管电泳法测定利巴韦林注射液的含量[J].中国医院药学杂志,2001(1):12-13
[14]韩加怡.利巴韦林及各中间体的高效毛细管电泳定量分析[J].药物分析杂志,2001,21(3):12-13.
[15]Roboz J,Suzuki R.Determination of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide(virazole)in blood and urine by chemical ionization-mass fragmentography[J].Journal of Chromatography A,1978,160(1):169-179.
[16]Li W,Luo S,Li S,et al.Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry[J].J Chroma-togr B,2007,846(1-2):57-58.
[17]Yeh L T,Nguyen M,Dadgostari S,et al.LC MS/MS method for simultaneous determination of viramidine and ribavirin level in monkey red blood cells[J].J Pharm Biomed Anal,2007,43(3):1057-1064.
[18]Liu Y F,Xu C,Yan R Z,et al.Sensitive and specific LC-MS/MS method for the simultaneous measurements of viramidine and ribavirin in human plasma[J].J Chroma-togr B,2006,832(1):17-23.
[19]Danso D,Langman L J,Snozek C L.LC MS/MS quantitation of ribavirin in serum and identification of endogenous isobaric interference[J].Clinica Chimica Acts,2011,412(23-24):2332-2335.
[20]曲斌,朱志谦,陆佳萍,等.测定肌肉中利巴韦林残留的固相萃取-UCLC-MS/MS法研究[J].中国家禽,2013,35(15):37-40.
[21]刘凯,王丽娜,李建忠.饲料和肌肉中利巴韦林的测定——超高效液相色谱-串联质谱法[J].现代畜牧兽医,2014(5):15-18.
[22]田光彩.利巴韦林片的微生物限度法验证试验[J].医药论坛杂志,2007,28(1):106.
[23]周剑,王敏,杨蒙瑞,等.抗病毒药物利巴韦林检测技术的研究进展[J].化学试剂,2016,38(1):1-6.
[24]李宇,王术皓,刘洪海,等.流动注射化学发光法测定利巴韦林[J].光谱实验室,2005,22(2):238-241.
[25]Henk VDL,Jan WCA,Wim TK,et al.Determination of ribavirin in human serum using liquid Chromatography tandem mass spectrometry[J].Talanta,2012,88:385-390.
[26]吴飞,丁黎.LC-MS法应用于利巴韦林颗粒剂的人体生物等效性研究[J].医学信息(中旬刊),2011(8):28-29.
[27]崔成富,陈创华,林海丹,等.中兽药剂中非法添加利巴韦林的检测方法研究[J].中国兽药杂志,2011(9):38-41.
[28]Zironi E,Gazzotti T,Lugoboni B,et al.Development of a rapid LC-MS/MS method for ribavirin determination in rat brain[J].Journal of Pharmaceutical & Biomedical Analysis,2011,54(4):889-892.
[29]朱永林,邵德佳,蒋天梅,等.高效液相色谱-串联质谱法测定鸡肝中利巴韦林及其代谢物残留总量[J].中国兽药杂志,2008,42(7):22-25.
[30]Shuang Peng,ShanshanSong,LiqiangLiu,et al.Rapid enzyme-linked immunosorbent assay and immunochromatographic strip for detecting ribavirin in chicken muscles[J].Food & Agricultural Immunology,2015,27(4):449-459.
[31]Zhaopeng Wang,Xuezhi Yu,Licai Ma,et al.Preparation of high affinity antibody for ribavirin with new haptens and residue analysis in chicken muscle,eggs and duck muscle[J].Food Additives and Contaminants Part A-Chemistry Analysis Control ment,2018,35(7):1247-1256.
[32]陈敏,崔海峰,冯才伟,等.利巴韦林单克隆抗体制备以及酶联免疫试剂盒的研究[J].中国酿造,2016,35(11):167-171.
[33]彭双.利巴韦林、金刚烷胺和氢氯噻嗪的免疫检测[D].无锡:江南大学,2016.
[34]王丽,桑亚新,周群标,等.大田软海绵酸人工抗原的合成及其多克隆抗体的制备[J].水产学报,2011,35(7):1007-1007.
[35]陈倩,陈荫楠,陈东海,等.基于单链抗体的呋喃唑酮酶联免疫检测方法的建立[J].食品科学,2017,38(20):242-247.
[36]Weilin L,Shelver,David J Smith.Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol[J].Journal of Agricultural & Food Chemistry,2004,52(8):2159-2166.
[37]周群标,桑亚新,王丽,等.动物性食品中盐酸克伦特罗ELISA检测方法的建立及应用[J].中国食品学报,2011,11(6):158-162.
[38]杜震环,井健.血蓝蛋白分子结构与生物学功能[J].生物过程,2015(3):30-37.
[39]郑小娇.动物性食品中氨苯砜免疫快速检测技术的研究[D].保定:河北农业大学,2015.
[2]谭胜国.因误用利巴韦林治理猪流感而引发药物中毒的诊疗报告[J].当代畜牧,2014(30):31-35.
[3]中华人民共和国农业部.中华人民共和国农业部公告第560号[Z].农业部公告[2005]560号.北京:农业部,2005-10-28.
[4]周剑,王敏,汤晓艳,等.畜禽产品监测用抗病毒类药物标准物质研制与应用情况[J].化学分析计量,2013,22(4):1-3.
[5]RK Austin,PE Trefts,M Hintz,et al.Sensitive radioimmunoassay for the broad-spectrum antiviral agent ribavirin[J].Antimicrobial Agents & Chemotherapy,1983,24(5):696-701.
[6]Melendez M,Rosario O,Zayas B,et al.HPLC-MS/MS method for the intracellular determination of ribavirin momophosphate and ribavirin triphosphate in CEMss cells[J].J Pharm Biomed Anal,2009,49(5):1233-240.
[7]D’Avolio A,Iba?nez A,Sciandra M,et al.Validation of liquid/liquid extraction method coupled with HPLC-UV for measurement of ribavirin plasma levels in HCV-positive patients[J].Journal of Chromatography B,2006,835(1):127-130.
[8]Homma M,Jayewardene A L,Gambertoglio J,et al.High-performance liquid chromatographic determination of ribavirin in whole blood to assess disposition in erythrocyte[J].Antimicrobial Agents and Chemotherapy,1999,43(11):2716-2719.
[9]Page T,Conor J D.The metabolism of ribavirin in erythrocytes and nucleated cells[J].International Journal of Biochemistry,1990,22(4):379-383.
[10]Berendsen B J A,Wegh R S,Essers M L,et al.Quantitative trace analysis of a broad range of antiviral drugs in poultry muscle using column-switch liquid chromatography coupled to tandem mass spectrometry[J].Analytical and Bioanalytical Chemistry,2012(4):1611-623.
[11]Zhang J Z,Zhao J,Zhou J H,et al.Determination of amantadine residue in honey by solid-phase extraction and high-performance liquid chromatography with pre-olumnderivatization and fluorometric detection[J].Chinese Journal of Chemistry,2011(8):1764-768.
[12]Michael C B,Regula T,Wolfgang T.Determination of ribavirin in human serum and plasma by capillary electrophoresis[J].Electrophoresis,2004,25(6):7-8.
[13]汪永忠,张国栋,吕布.高效毛细管电泳法测定利巴韦林注射液的含量[J].中国医院药学杂志,2001(1):12-13
[14]韩加怡.利巴韦林及各中间体的高效毛细管电泳定量分析[J].药物分析杂志,2001,21(3):12-13.
[15]Roboz J,Suzuki R.Determination of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide(virazole)in blood and urine by chemical ionization-mass fragmentography[J].Journal of Chromatography A,1978,160(1):169-179.
[16]Li W,Luo S,Li S,et al.Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry[J].J Chroma-togr B,2007,846(1-2):57-58.
[17]Yeh L T,Nguyen M,Dadgostari S,et al.LC MS/MS method for simultaneous determination of viramidine and ribavirin level in monkey red blood cells[J].J Pharm Biomed Anal,2007,43(3):1057-1064.
[18]Liu Y F,Xu C,Yan R Z,et al.Sensitive and specific LC-MS/MS method for the simultaneous measurements of viramidine and ribavirin in human plasma[J].J Chroma-togr B,2006,832(1):17-23.
[19]Danso D,Langman L J,Snozek C L.LC MS/MS quantitation of ribavirin in serum and identification of endogenous isobaric interference[J].Clinica Chimica Acts,2011,412(23-24):2332-2335.
[20]曲斌,朱志谦,陆佳萍,等.测定肌肉中利巴韦林残留的固相萃取-UCLC-MS/MS法研究[J].中国家禽,2013,35(15):37-40.
[21]刘凯,王丽娜,李建忠.饲料和肌肉中利巴韦林的测定——超高效液相色谱-串联质谱法[J].现代畜牧兽医,2014(5):15-18.
[22]田光彩.利巴韦林片的微生物限度法验证试验[J].医药论坛杂志,2007,28(1):106.
[23]周剑,王敏,杨蒙瑞,等.抗病毒药物利巴韦林检测技术的研究进展[J].化学试剂,2016,38(1):1-6.
[24]李宇,王术皓,刘洪海,等.流动注射化学发光法测定利巴韦林[J].光谱实验室,2005,22(2):238-241.
[25]Henk VDL,Jan WCA,Wim TK,et al.Determination of ribavirin in human serum using liquid Chromatography tandem mass spectrometry[J].Talanta,2012,88:385-390.
[26]吴飞,丁黎.LC-MS法应用于利巴韦林颗粒剂的人体生物等效性研究[J].医学信息(中旬刊),2011(8):28-29.
[27]崔成富,陈创华,林海丹,等.中兽药剂中非法添加利巴韦林的检测方法研究[J].中国兽药杂志,2011(9):38-41.
[28]Zironi E,Gazzotti T,Lugoboni B,et al.Development of a rapid LC-MS/MS method for ribavirin determination in rat brain[J].Journal of Pharmaceutical & Biomedical Analysis,2011,54(4):889-892.
[29]朱永林,邵德佳,蒋天梅,等.高效液相色谱-串联质谱法测定鸡肝中利巴韦林及其代谢物残留总量[J].中国兽药杂志,2008,42(7):22-25.
[30]Shuang Peng,ShanshanSong,LiqiangLiu,et al.Rapid enzyme-linked immunosorbent assay and immunochromatographic strip for detecting ribavirin in chicken muscles[J].Food & Agricultural Immunology,2015,27(4):449-459.
[31]Zhaopeng Wang,Xuezhi Yu,Licai Ma,et al.Preparation of high affinity antibody for ribavirin with new haptens and residue analysis in chicken muscle,eggs and duck muscle[J].Food Additives and Contaminants Part A-Chemistry Analysis Control ment,2018,35(7):1247-1256.
[32]陈敏,崔海峰,冯才伟,等.利巴韦林单克隆抗体制备以及酶联免疫试剂盒的研究[J].中国酿造,2016,35(11):167-171.
[33]彭双.利巴韦林、金刚烷胺和氢氯噻嗪的免疫检测[D].无锡:江南大学,2016.
[34]王丽,桑亚新,周群标,等.大田软海绵酸人工抗原的合成及其多克隆抗体的制备[J].水产学报,2011,35(7):1007-1007.
[35]陈倩,陈荫楠,陈东海,等.基于单链抗体的呋喃唑酮酶联免疫检测方法的建立[J].食品科学,2017,38(20):242-247.
[36]Weilin L,Shelver,David J Smith.Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol[J].Journal of Agricultural & Food Chemistry,2004,52(8):2159-2166.
[37]周群标,桑亚新,王丽,等.动物性食品中盐酸克伦特罗ELISA检测方法的建立及应用[J].中国食品学报,2011,11(6):158-162.
[38]杜震环,井健.血蓝蛋白分子结构与生物学功能[J].生物过程,2015(3):30-37.
[39]郑小娇.动物性食品中氨苯砜免疫快速检测技术的研究[D].保定:河北农业大学,2015.