呋喃它酮代谢物时间分辨荧光免疫分析法的建立与应用
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摘要
建立了Eu~(3+)标记的间接竞争时间分辨荧光免疫分析法(Indirect competitive time-resolved fluoroimmunoassay,ic-TRFIA)用于鱼肉样品中呋喃它酮代谢物AMOZ的检测。通过单因素实验考察了包被抗原浓度、抗体稀释倍数、竞争反应时间等参数对方法灵敏度的影响。结果表明,ic-TRFIA的最佳反应条件为:包被抗原浓度为0.25μg/m L,抗体稀释5×10~4倍,最佳竞争时间为50 min。在优化的条件下,方法的检出限(LOD,IC_(10))为0.01 ng/m L,半抑制浓度(IC_(50))为0.26 ng/m L,线性范围(IC_(20)~IC_(80))为0.025~2.83 ng/m L,对鱼样中AMOZ回收率在78.0%~86.0%之间,各样品变异系数均小于15%,与HPLC-MS/MS对比检测结果显示相关性良好。本方法灵敏度高、特异性好,能够满足实际样品的检测需求。
To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone( AMOZ) in fish sample,an Eu~(3+)labeling time-resolved fluoroimmunoassay( TRFIA) was developed. The effects of experimental conditions including AMOZA-OVA concentration,dilution of antibody,and reaction time on the sensitivity of TRFIA were explored. The results showed that the optimized assay conditions were as follows: the AMOZA-OVA concentration was 0. 25 μg / m L; the antibody was diluted 5 × 10~4 folds,and the competitive reaction time was 50 min. Under optimal conditions,the method showed a detection limit of 0. 01 ng / m L,an IC_(50) of 0. 26 ng / m L and a linear range( IC_(20)-IC_(80)) of 0. 025- 2. 83 ng /m L. The recoveries of AMOZ in fish at three spiked levels ranged from 78. 0% to 86. 0%,and the relative standard deviations were less than15%. Good correlation between the ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suited for the determination of AMOZ residue in food samples.
To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone( AMOZ) in fish sample,an Eu~(3+)labeling time-resolved fluoroimmunoassay( TRFIA) was developed. The effects of experimental conditions including AMOZA-OVA concentration,dilution of antibody,and reaction time on the sensitivity of TRFIA were explored. The results showed that the optimized assay conditions were as follows: the AMOZA-OVA concentration was 0. 25 μg / m L; the antibody was diluted 5 × 10~4 folds,and the competitive reaction time was 50 min. Under optimal conditions,the method showed a detection limit of 0. 01 ng / m L,an IC_(50) of 0. 26 ng / m L and a linear range( IC_(20)-IC_(80)) of 0. 025- 2. 83 ng /m L. The recoveries of AMOZ in fish at three spiked levels ranged from 78. 0% to 86. 0%,and the relative standard deviations were less than15%. Good correlation between the ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suited for the determination of AMOZ residue in food samples.
引文
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15 DAI Jin-Bo,XU Zhen-Lin,LIU Feng-Yin,YANG Jin-Yi,SUN Yuan-Ming,WANG Hong,LEI Hong-Tao,SHEN YuDong.Chinese J.Anal.Chem.,2015,43(6):871-875戴尽波,徐振林,刘凤银,杨金易,孙远明,王弘,雷红涛,沈玉栋.分析化学,2015,43(6):871-875
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18 Chen J,Liu T,Chen Z,Hou J,Wu Y.J.Med.Virol.,2015,87(11):1940-1945
19 Zhang Z,Liu J F,Feng T T,Yao Y,Gao L H,Jiang G B.Environ.Sci.Technol.,2013,47(1):454-462
20 Shi H Y,Sheng E Z,Feng L,Zhou L L,Hua X D,Wang M H.Environ.Sci.Pollut.R.,2015,22(19):14882-14890
21 JIN Jing,LAI Wei-Hua,TU Zu-Xin,LI Peng.Food Sci.,2006,27(12):886-889金晶,赖卫华,涂祖新,李鹏.食品科学,2006,27(12):886-889
22 Zhang J,Gao L,Zhou B,Zhu L,Zhang Y,Huang B A.J.Agr.Food Chem.,2011,91(2):193-197
23 GB/T 18932.24-2005,Method for the Determination of the Metabolite Residues of Furaltadon,Nitrofurazone,Nitrofurantoin and furazolidone in Honey-Liquid Chromatography-Tandem Mass Spectrometry Method.National Standards of the People's Republic of China蜂蜜中吠喃它酮、吠喃西林、吠喃妥因和吠喃哇酮代谢物残留量的测定方法液相色谱-串联质谱法.中华人民共和国国家标准.GB/T 18932.24-2005
2 Bartel L C,Montalto de M M,Castro J A.Food Chem.Toxicol.,2009,47(1):140-144
3 O'Mahony J,Moloney M,Mc Connell R I,Benchikh E O,Lowry P,Furey A,Danaher M.Biosens.Bioelectron.,2011,26(10):4076-4081
4 ZOU Long,LIU Shi-Wen,XU Yang.Sci.Technol.Food Ind.,2011,32(9):464-471邹龙,刘师文,许杨.食品工业科技,2011,32(9):464-471
5 CHEN Wei-Feng,CHEN Jing-Xin.Meat Research,2011,25(12):53-57陈威风,陈敬鑫.肉类研究,2011,25(12):53-57
6 Barbosa J,Moura S,Barbosa R,Ramos F,da Silveira M I N.Anal.Chim.Acta,2007,586(1-2):359-365
7 Noonan G O,Warner C R,Hsu W,Begley T H,Perfetti G A,Diachenko G W.J.Agr.Food Chem.,2005,53(12):4680-4685
8 Leston S,Nunes M,Freitas A,Barbosa J,Ramos F,Pardal M A.J.Chromatogr.B,2011,879(32):3832-3836
9 Barbosa J,Freitas A,Moura S,Mourao JL,da Silveira M I N,Ramos F.J.Agr.Food Chem.,2011,59(22):11927-11934
10 Barbosa J,Freitas A,Mourao JL,da Silveira M I N,Ramos F.J.Agr.Food Chem.,2012,60(17):4227-4234
11 Shen Y D,Xu Z L,Zhang S W,Wang H,Yang J Y,Lei H T,Xiao Z L,Sun Y M.J.Agr.Food Chem.,2012,60(44):10991-10997
12 Xu Z L,Shen Y D,Sun Y M,Campbell K,Tian Y X,Zhang S W,Lei H T,Jiang Y M.Talanta,2013,103:306-313
13 Jester ELE,Abraham A,Wang Y S,El Said K R,Plakas S M.Food Chem.,2014,145:593-598
14 Liu Y C,Jiang W,Chen Y J,Xiao Y,Shi J L,Qiao Y B,Zhang H J,Li T,Wang Q.J.Immunol.Methods,2013,395(1-2):29-36
15 DAI Jin-Bo,XU Zhen-Lin,LIU Feng-Yin,YANG Jin-Yi,SUN Yuan-Ming,WANG Hong,LEI Hong-Tao,SHEN YuDong.Chinese J.Anal.Chem.,2015,43(6):871-875戴尽波,徐振林,刘凤银,杨金易,孙远明,王弘,雷红涛,沈玉栋.分析化学,2015,43(6):871-875
16 QIAO Zi-Wen,WANG Hai-Yu,WANG Lei.Chinese J.Lumin.,2014,35(7):791-794乔自文,王海宇,王雷.发光学报,2014,35(7):791-794
17 HU Li-Hua,ZHOU Di,ZHU Jian,HUANG Zhi-Bin.Chemical Research and Application,2010,22(8):969-974胡丽华,周弟,朱健,黄志斌.化学研究与应用.2010,22(8):969-974
18 Chen J,Liu T,Chen Z,Hou J,Wu Y.J.Med.Virol.,2015,87(11):1940-1945
19 Zhang Z,Liu J F,Feng T T,Yao Y,Gao L H,Jiang G B.Environ.Sci.Technol.,2013,47(1):454-462
20 Shi H Y,Sheng E Z,Feng L,Zhou L L,Hua X D,Wang M H.Environ.Sci.Pollut.R.,2015,22(19):14882-14890
21 JIN Jing,LAI Wei-Hua,TU Zu-Xin,LI Peng.Food Sci.,2006,27(12):886-889金晶,赖卫华,涂祖新,李鹏.食品科学,2006,27(12):886-889
22 Zhang J,Gao L,Zhou B,Zhu L,Zhang Y,Huang B A.J.Agr.Food Chem.,2011,91(2):193-197
23 GB/T 18932.24-2005,Method for the Determination of the Metabolite Residues of Furaltadon,Nitrofurazone,Nitrofurantoin and furazolidone in Honey-Liquid Chromatography-Tandem Mass Spectrometry Method.National Standards of the People's Republic of China蜂蜜中吠喃它酮、吠喃西林、吠喃妥因和吠喃哇酮代谢物残留量的测定方法液相色谱-串联质谱法.中华人民共和国国家标准.GB/T 18932.24-2005