牛奶及水样中泰乐菌素酶联免疫检测方法研究
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摘要
针对环境及食品中泰乐菌素残留问题,本研究将泰乐菌素分子结构侧链醛基一步直接氧化为羧基,并将其作为新的半抗原,通过免疫新西兰大白兔制备了特异性的抗泰勒菌素多克隆抗体,优化的反应条件为:包被原稀释6000倍,抗体稀释2000倍,反应缓冲液为pH 7.4、吐温含量0.1%、离子浓度0.01 mol/L的PBST,二抗稀释倍数为4000倍,二抗反应时间30 min。在此优化条件下,建立了检测泰乐菌素的间接竞争酶联免疫分析法(icELISA),半数抑制浓度(IC_(50))为1.39 ng/mL,线性范围(IC_(20)~IC_(80))为0.17~11.0 ng/mL,检出限(IC_(10))为0.07 ng/mL,与结构功能类似物交叉反应率均小于0.1%。对纯牛奶、自来水、鱼塘水中泰乐菌素的添加回收率在78.4%~105.6%之间,RSD<15%,检测结果与HPLC-MS/MS方法具有良好的相关性(R~2=0.97)。本研究建立的icELISA方法适用于牛奶及水样中泰乐菌素残留的特异性快速筛查。
To address the issues of tylosin residues in food and environment,a good specific polyclonal antibody was prepared by animal immunization based on a new hapten achieved by oxidizing the aldehyde group on the side chain of tylosin to carboxyl. Further,an indirect competitive enzyme-linked immunosorbent assay( icELISA) was established. The main experimental conditions were as follows: the coating antigen and the antibody were respectively diluted by 6000 times and 2000 times, the reaction buffer was PBST( pH 7.4),and the Tween concentration and ionic concentration were 0.1% and 0.01 mol/L,respectively.The dilution ratio of the secondary antibody was 4000 times and the secondary reaction time was 30 min. Under the optimized conditions, the IC_(50) and the linear range of this method were 1. 39 ng/mL and 0. 17-11.0 ng/mL,respectively. The detection limit was 0. 07 ng/mL,and the cross-reactivity with analogues was less than 0.1%. The recoveries of spiked raw milk and water samples were 78. 4%-105. 6% with relative standard deviation less than 15%. Besides,the detection results had good correlation with HPLC method( R~2= 0.97). This method is suitable for rapid and specific screening of tylosin residues in milk and water samples.
To address the issues of tylosin residues in food and environment,a good specific polyclonal antibody was prepared by animal immunization based on a new hapten achieved by oxidizing the aldehyde group on the side chain of tylosin to carboxyl. Further,an indirect competitive enzyme-linked immunosorbent assay( icELISA) was established. The main experimental conditions were as follows: the coating antigen and the antibody were respectively diluted by 6000 times and 2000 times, the reaction buffer was PBST( pH 7.4),and the Tween concentration and ionic concentration were 0.1% and 0.01 mol/L,respectively.The dilution ratio of the secondary antibody was 4000 times and the secondary reaction time was 30 min. Under the optimized conditions, the IC_(50) and the linear range of this method were 1. 39 ng/mL and 0. 17-11.0 ng/mL,respectively. The detection limit was 0. 07 ng/mL,and the cross-reactivity with analogues was less than 0.1%. The recoveries of spiked raw milk and water samples were 78. 4%-105. 6% with relative standard deviation less than 15%. Besides,the detection results had good correlation with HPLC method( R~2= 0.97). This method is suitable for rapid and specific screening of tylosin residues in milk and water samples.
引文
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14 BAO Jie-Hua,ZHANG Jin-Xia,GUO Qiang-Gong,ZHANG Ping.Chinese Journal of Veterinary Drug,2015,49(2):35-39包洁华,张金霞,郭强功,张萍.中国兽药杂志,2015,49(2):35-39
15 CAO Ai-Ying,ZHANG Yuan,ZHAO Chun-Hui,WANG Xiu-Lin,PAN Yong,LI Ying,ZONG Chao,CAO Jie.Chinese Fishery Quality and Standards,2013,3(4):21-24曹爱英,张园,赵春晖,王秀林,潘勇,李英,宗超,曹洁.中国渔业质量与标准,2013,3(4):21-24
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18 Molognoni L,Valese A C,Lorenzetti A,Daguer H,Lindner J D D.Food Chem.,2016,211:748-756
19 van den Meersche T,van Pamel E,van Poucke C,Herman L,Heyndrickx M,Rasschaert G,Daeseleire E.J.Chromatogr.A,2016,1429:248-257
20 Zhang J K,Qi Y H,Liu J X,Wang J P.Food Agric.Immunol.,2013,24(4):419-431
21 Le T,He H,Niu X,Chen Y Xu J.Food Agric.Immunol.,2013,24(4):467-480
22 Li X,Shen J,Wang Q,Gao S,Pei X,jiang H,Wen K.Anal.Bioanal.Chem.,2015,407(30):9125-9133
23 Zhang J,Liu J,Wang L,Lin Chai,Wang J.J.Environ.Sci.Heal.B,2012,47(9):876-882
24 Song Y,Song S,Liu L,Kuang H,Guo L,Xu C.Food Agric.Immunol.,2015,27(3):314-328
25 Chen Z J,Fu H J,Luo L,Sun Y M,Yang J Y,Zeng D P,Shen Y D,Xu Z L.Anal.Methods,2017,9(10):1617-1626
26 Zhang Y Q,Wang L T,Shen X,Wei X Q,Huang X A,Liu Y J,Sun X L,Wang Z H,Sun Y M,Xu Z L,Eremin S A,Lei H T.J.Agric.Food Chem.,2017,65(23):4830-4838
27 Wu J,Shen Y D,Lei H T,Sun Y M,Yang J Y,Xiao Z L,Wang H,Xu Z L.J.Agric.Food Chem.,2014,62(29):7078-7084
28 Luo L,Xu Z L,Yang J Y,Xiao Z L,Li Y J,Beier R C,Sun Y M,Lei H T,Wang H,Shen Y D.J.Agric.Food Chem.,2014,62(51):12299-12308
29 Cai X,Whitfield T,Moreno A Y,Grant Y,Hixon M S,Koob G F,Janda K D.Mol.Pharm.,2013,10(11):4176-4184
30 Zhang W,He L,Zhang R,Guo S,Yue H,Ning X,Tan G,Li Q X,Wang B.Food Chem.,2016,207:233-238
31 GB/T 22988-2003,Determination of Spiramycin,Pyrimidin,Oleandomycin,Tilmicosin,Erythromycin,and Tylosin in Milk and Milk Powder by Liquid Chromatography-Tandem Mass Spectrometry.National Standards of the People's Republic of China牛奶和奶粉中螺旋霉素、吡利霉素、竹桃霉素、替米考星、红霉素、泰乐菌素残留量的测定——液相色谱-串联质谱法.中华人民共和国国家标准.GB/T 22988-2003
2 Stephens T P,Stanford K,Mcallister T A.Can.J.Anim.Sci.,2009,89(1):167-167
3 SHEN Xiang-Rong,SU Ya-Nan,LIU Cai,YANG Di,ZHANG Xiao-Hui,ZONG Xin-Ru,WANG Wen-Chao,ZHANG Miao,ZHANG Xu,BAO En-Dong.Journal of Nanjing Agricultural University,2013,5:120-124沈向荣,苏亚楠,刘彩,杨迪,张晓辉,宗欣茹,王文超,张淼,张栩,鲍恩东.南京农业大学,2013,5:120-124
4 LI Hong-Sheng,ZHANG Ji-Yu,ZHOU Xu-Zheng,LI Jian-Yong,LI Jin-Shan,MIAO Xiao-Lou,ZHANG Zhan-Feng.Progress In Veterinary Medicine,2006,27(2):94-96李宏胜,张继瑜,周绪正,李剑勇,李金善,苗小楼,张占峰.动物医学进展,2006,27(2):94-96
5 Hume M E,Donskey C J.Foodborne Pathog.Dis.,2017,14(4):231-237
6 Gautierbouchardon A V,Reinhardt A K,Kobisch M,Kempf I.Vet.Microbiol.,2002,88(1):47-58
7 CHEN Zhang-Liu,WU Cong-Ming,JIANG Hong-Xia,LIAO Xiao-Ping,WEI Xiu-Li,DING Huan-Zhong,ZENG ZhenLing.Sichuan Journal of Physiological Sciences,2003,25(3):120-123陈杖榴,吴聪明,蒋红霞,廖晓萍,魏秀丽,丁焕中,曾振灵.四川生理科学杂志,2003,25(3):120-123
8 Council Regulation(EC)No.37/2010.Pharmacologically Active Substances and Their Classification Regarding Maximum Residue Limits in Foodstuffs of Animal Origin.European Commission
9 Animal Origin Food Veterinary Drug Maximum Residue Limit.Ministry of Agriculture Bulletin[2002]No.235动物源食品中兽药最高残留限量.农业部公告[2002]第235号
10 Garder J L,Moorman T B,Soupir M L.J.Environ.Qual.,2014,43(4):1484-1493
11 Luby E M,Moorman T B,Soupir M L.Sci.Total Environ.,2016,550:1126-1133
12 HUANG Chen,CHEN Ben-Long,WANG Nai-Fu,LI Wei-Hua.China Animal Health Inspection,2016,33(2):29-33黄晨,陈本龙,王乃福,李卫华.中国动物检疫,2016,33(2):29-33
13 Chebira B,Boultif L,Agabou A,Mekroud A.Adv.Anim.Vet.Sci.,2015,3(5):295-301
14 BAO Jie-Hua,ZHANG Jin-Xia,GUO Qiang-Gong,ZHANG Ping.Chinese Journal of Veterinary Drug,2015,49(2):35-39包洁华,张金霞,郭强功,张萍.中国兽药杂志,2015,49(2):35-39
15 CAO Ai-Ying,ZHANG Yuan,ZHAO Chun-Hui,WANG Xiu-Lin,PAN Yong,LI Ying,ZONG Chao,CAO Jie.Chinese Fishery Quality and Standards,2013,3(4):21-24曹爱英,张园,赵春晖,王秀林,潘勇,李英,宗超,曹洁.中国渔业质量与标准,2013,3(4):21-24
16 Jank L,Martins M T,Arsand J B,Motta M C,Hoff R B,Barreto F,Pizzolato T M.Talanta,2015,144:686-695
17 XU Jie,DENG Chao,XIAN Qi-Ming.China Mesurement and Test,2017,43(3):58-62徐洁,邓超,鲜啟鸣.中国测试,2017,43(3):58-62
18 Molognoni L,Valese A C,Lorenzetti A,Daguer H,Lindner J D D.Food Chem.,2016,211:748-756
19 van den Meersche T,van Pamel E,van Poucke C,Herman L,Heyndrickx M,Rasschaert G,Daeseleire E.J.Chromatogr.A,2016,1429:248-257
20 Zhang J K,Qi Y H,Liu J X,Wang J P.Food Agric.Immunol.,2013,24(4):419-431
21 Le T,He H,Niu X,Chen Y Xu J.Food Agric.Immunol.,2013,24(4):467-480
22 Li X,Shen J,Wang Q,Gao S,Pei X,jiang H,Wen K.Anal.Bioanal.Chem.,2015,407(30):9125-9133
23 Zhang J,Liu J,Wang L,Lin Chai,Wang J.J.Environ.Sci.Heal.B,2012,47(9):876-882
24 Song Y,Song S,Liu L,Kuang H,Guo L,Xu C.Food Agric.Immunol.,2015,27(3):314-328
25 Chen Z J,Fu H J,Luo L,Sun Y M,Yang J Y,Zeng D P,Shen Y D,Xu Z L.Anal.Methods,2017,9(10):1617-1626
26 Zhang Y Q,Wang L T,Shen X,Wei X Q,Huang X A,Liu Y J,Sun X L,Wang Z H,Sun Y M,Xu Z L,Eremin S A,Lei H T.J.Agric.Food Chem.,2017,65(23):4830-4838
27 Wu J,Shen Y D,Lei H T,Sun Y M,Yang J Y,Xiao Z L,Wang H,Xu Z L.J.Agric.Food Chem.,2014,62(29):7078-7084
28 Luo L,Xu Z L,Yang J Y,Xiao Z L,Li Y J,Beier R C,Sun Y M,Lei H T,Wang H,Shen Y D.J.Agric.Food Chem.,2014,62(51):12299-12308
29 Cai X,Whitfield T,Moreno A Y,Grant Y,Hixon M S,Koob G F,Janda K D.Mol.Pharm.,2013,10(11):4176-4184
30 Zhang W,He L,Zhang R,Guo S,Yue H,Ning X,Tan G,Li Q X,Wang B.Food Chem.,2016,207:233-238
31 GB/T 22988-2003,Determination of Spiramycin,Pyrimidin,Oleandomycin,Tilmicosin,Erythromycin,and Tylosin in Milk and Milk Powder by Liquid Chromatography-Tandem Mass Spectrometry.National Standards of the People's Republic of China牛奶和奶粉中螺旋霉素、吡利霉素、竹桃霉素、替米考星、红霉素、泰乐菌素残留量的测定——液相色谱-串联质谱法.中华人民共和国国家标准.GB/T 22988-2003