沙拉沙星间接竞争化学发光酶联免疫检测方法的建立

doi:10.16431/j.cnki.1671-7236.2021.05.029

Abstract

Abstract: To establish a rapid detection method for sarafloxacin (SAR) drug residues in animal-derived foods,SAR monoclonal antibody was prepared by immunizing mice with sarafloxacin hydrochloride molecular modification and coupling protein synthesis complete immunogen.The checkerboard method was used to determine the optimal coating concentration (SAR-1-OVA) and primary antibody dilution,and optimize the optimal reaction conditions to establish an indirect chemiluminescence enzyme-linked immunoassay (ic-CLEIA).The method was evaluated by sensitivity,precision,cross-reaction rate,and additive recovery test.The results showed that the complete immunogen coupling was successful after UV scanning,and the prepared SAR monoclonal antibody titer reached 1:4×10⁶.The optimal reaction conditions of ic-CLEIA were as follows:Coating concentration was 0.25 μg/mL,dilution of antibody was 1:750 000,coating at 4 ℃ overnight,5% skim milk sealed,competitive incubation for 1 h,dilution of enzyme labeled secondary antibody was 1:10 000,incubation for 1 h.The standard curve was linear,the linear range was 0.0625 to 10 ng/mL,R²=0.995,the sensitivity IC₅₀ was 1.45 ng/mL.The average intra- and inter-assay coefficients of variation were both <10%.Except the cross reaction rate with difloxacin was 98.08%,the cross reaction rates with other fluoroquinolones were <8%,and there was no cross reaction with other non fluoroquinolones.The minimum detection limit (LOD) of standard SAR was 0.32 ng/mL,and the LOD of SAR in chicken samples was 0.46 μg/kg.The recovery rate of standard addition was in the range of 88.3% to 106.7%,and the coefficient of variation was ≤12.2%.The above results showed that the method was fast and sensitive,was suitable for the large-scale detection of sarafloxacin residues in animal-derived foods,and this results provided a new method for the detection of sarafloxacin residues.

Key words: sarafloxacin (SAR); monoclonal antibody; indirect competitive chemiluminescence enzyme-linked immunoassay (ic-CLEIA); residue detection

CLC Number:

S859.84

LI Si, JIANG Wei, CHENG Tingting, LU Chunhui, XIN Sipei, CAO Jingzheng, WANG Quan, SUN Weidong. Establishment of an Indirect Competitive Chemiluminescence Enzyme-linked Immunoassay for Sarafloxacin[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1772-1783.

References

[1] 邱银生,吴佳.动物专用氟喹诺酮类药物研究进展简介[J].中国兽药杂志,1998,3:46-48. QIU Y S,WU J.Introduction to the research progress of fluoroquinolones for animals[J].Chinese Journal of Veterinary Drug,1998,3:46-48.(in Chinese)
[2] 李艳华,闫清波,孙丁岩,等.兽用喹诺酮类抗菌剂沙拉沙星的研究概况[J].黑龙江畜牧兽医,2001,9:29-31. LI Y H,YAN Q B,SUN D Y,et al.Research overview of veterinary quinolone antibacterial agent sarafloxacin[J].Heilongjiang Animal Science and Veterinary Medicine,2001,9:29-31.(in Chinese)
[3] WANG H W,LIU Y Q,WEI S L,et al.Selective extraction and determination of fluoroquinolones in bovine milk samples with montmorillonite magnetic molecularly imprinted polymers and capillary electrophoresis[J].Analytical and Bioanalytical Chemistry,2016,408(2):589-598.
[4] 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. ZHANG D D,SU C,HONG W,et al.Research progress on detection of fluoroquinolones residues in edible animal products[J].Anhui Agricultural Science Bulletin,2016,22(13):38-41.(in Chinese)
[5] 杨长志,刘永,孟冰冰,等.动物源性食品中五种氟喹诺酮类药物残留量的同时测定[J].分析试验室,2008,27(9):82-85. YANG C Z,LIU Y,MENG B B,et al.Simultaneous determination of five fluoroquinolones residues in foodstuff of animal origin[J].Chinese Journal of Analysis Laboratory,2008,27(9):82-85.(in Chinese)
[6] LUI N,ZHAO Z Y,TAN Y L,et al.Simultaneous raising of rabbit monoclonal antibodies to fluoro-quinolones with diverse recognition functionalities via single mixture immunization[J].Analytical Chemistry,2016,88(2):1246-1252.
[7] 刘丁溪,杨杰程,周宏超,等.动物性食品中抗菌药物残留检测方法研究进展[J].中国畜牧兽医,2019,46(7):2183-2192. LIU D X,YANG J C,ZHOU H C,et al.Research advance on detection methods of antibacterial residues in animal food[J].China Animal Husbandry & Veterinary Medicine,2019,46(7):2183-2192.(in Chinese)
[8] 王勇,龚勇,卢明华.氟喹诺酮类药物残留检测方法的研究进展[J].中国畜牧兽医,2017,44(8):2509-2516. WANG Y,GONG Y,LU M H.Research progress on detection methods of fluoroquinones residues[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2509-2516.(in Chinese)
[9] OKENMAN L,VAN H J,DEBEUCKELAERE W.Evaluation of the European four-plate test as a tool for screening antibiotic residues in meat samples from retail outlets[J].Journal of Aoac International,1998,81(1):51-56.
[10] 王蕊,廖晓萍,刘雅红.氟喹诺酮类兽药残留检测方法研究进展[J].广东农业科学,2010,10:41-43. WANG R,LIAO X P,LIU Y H.Research progress of fluoroquinolone veterinary drug residue detection methods[J].Guangdong Agricultural Sciences,2010,10:41-43.(in Chinese)
[11] 乔坤云,孙涛,刘圣红,等.高效液相色谱荧光法检测鸡肉中4种氟喹诺酮药物残留[J].分析仪器,2015,6:43-46. QIAO K Y,SUN T,LIU S H,et al.Detection of 4 fluoroquinolone drug residues in chicken by high performance liquid chromatography with fluore-scence[J].Analytical Instrumentation,2015,6:43-46.(in Chinese)
[12] 李雷霆,刘均华,刘宝涛,等.高效液相色谱紫外法同时检测鸡蛋中5种青霉素类药物残留的研究[J].中国畜牧兽医,2017,44(8):2483-2488. LI L T,LIU J H,LIU B T,et al.Residue analysis of five kinds of penicillins in eggs by high performance liquid chromatography-ultraviolet detection[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2483-2488.(in Chinese)
[13] HE X,WANG G N,YANG K,et al.Magnetic graphene dispersive solid phase extraction combining high performance liquid chromatography for determi-nation of fluoroquinolones in foods[J].Food Chemistry,2017,221:1226-1231.
[14] 杨卫军,李曼,曹秀梅,等.超高效液相色谱法检测鸡肉中氟喹诺酮类药物残留[J].现代畜牧兽医,2016,4:8-12. YANG W J,LI M,CAO X M,et al.Discussion on determination of fluoroquinolones residues in chicken by ultra-performance liquid chromatography[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2016,4:8-12.(in Chinese)
[15] PEARCE J N,BUMS B G,RIET J M,et al.Determination of fluoroquinolones in aquaculture products by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)[J].Food Additives & Contaminants. Part A,Chemistry,Analysis,Control,Exposure & Risk Assessment,2009,26(1):39-46.
[16] MENG Z,SHI Z,LIANG S,et al.Residues investigation of fluoroquinolones and sulphonamides and their metabolites in bovine milk by quantification and confirmation using ultra-performance liquid chromatography-tandem mass spectrometry[J].Food Chemistry,2015,174:597-605.
[17] TAO X,CHEN M,JIANG H,et al.Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment[J].Analytical and Bioanalytical Chemistry,2013,405(23):7477-7484.
[18] 何方洋,冯月君,吴鹏,等.氯霉素化学发光酶联免疫检测方法的建立[J].中国兽药杂志,2012,46(3):25-29. HE F Y,FENG Y J,WU P,et al.Establishment of chemiluminescent enzyme immunoassay for chloram-phenicol[J].Chinese Journal of Veterinary Drug, 2012,46(3):25-29.(in Chinese)
[19] 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. XU R.Gold immunochromatography technology for the rapid detection of the enrofloxacin in seafood[D].Qingdao:Ocean University of China,2013.(in Chinese)
[20] 陈新建,孟繁静.玉米赤霉烯酮的固相放射免疫分析[J].植物生理学通讯,1994,2:121-124. CHEN X J,MENG F J.Solid-phase radioimmunoassay (RIA) for zearalenone[J].Plant Physiology Commu-nications,1994,2:121-124.(in Chinese)
[21] LIU Y Z,ZHAO G X,LIU J,et al.Synthesis of novel haptens against ciprofloxacin and production of generic monoclonal antibodies for immunoscreening of fluoroquinolones in meat[J].Journal of the Science of Food and Agriculture,2013,93(6):1370-1377.
[22] YAO C,XU Z,WANG H,et al.High affinity antibody based on a rationally designed hapten and development of a chemiluminescence enzyme immu-noassay for quantification of alternariol in fruit juice,maize and flour[J].Food Chemistry,2019,283:359-366.
[23] 马震原,王淑娟,闫若潜,等.猪伪狂犬病病毒gB蛋白抗体竞争化学发光酶联免疫检测方法的建立[J].畜牧兽医学报,2020,51(3):574-583. MA Z Y,WANG S J,YAN R Q,et al.Establishment of a competitive chemiluminescent enzyme immunoassay for detection antibodies aganist gB protein of Pseudorabies virus[J].Chinese Journal of Animal and Veterinary Sciences,2020,51(3):574-583.(in Chinese)
[24] 熊挺,蒋蔚,韩阳瑞,等.家禽饲料中玉米赤霉烯酮化学发光酶联免疫分析方法的建立[J].畜牧与兽医,2018,50(2):122-130. XIONG T,JIANG W,HAN Y R,et al.Development of chemiluminescence enzyme immunoassays for determining zearalenone in poultry feed[J].Animal Husbandry and Veterinary Medicine,2018,50(2):122-130.(in Chinese)
[25] LIU Y C,JIANG W,CHEN Y J,et al.A novel chemiluminescent ELISA for detecting furaltadone metabolite,3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) in fish,egg,honey and shrimp samples[J].Journal of Immunological Methods,2013,395(1-2):29-36.
[26] 宫秀杰,萨仁高娃,阿力腾才斯克,等.牛奶中氟喹诺酮类药物残留的HPLC检测方法研究[J].草食家畜,2020,4:57-62. GONG X J,SARENGAOWA,ALITENGCAISIKE,et al.Determination of residues of fluroquinolones in milk by HPLC[J].Grass-Feeding Livestock,2020,4:57-62.(in Chinese)
[27] 刘儒彪,胡骁飞,张改平,等.沙拉沙星ELISA快速检测试剂盒的研制及性能鉴定[J].食品与发酵工业,2015,41(2):189-194. LIU R B,HU X F,ZHANG G P,et al.Development and application of rapid test ELISA kit in sarafloxacin[J]. Food and Fermentation Industries,2015,41(2):189-194.(in Chinese)
[28] 孙晓峥,任柯潼,胡叶军,等.肉鸡组织中氟喹诺酮类抗生素快速检测的胶体金技术研究[J].黑龙江畜牧兽医,2018,19:208-213. SUN X Z,REN K T,HU Y J,et al.Colloidal gold technology for rapid detection of fluoroquinolone antibiotics in broiler tissues[J].Heilongjiang Animal Science and Veterinary Medicine,2018,19:208-213.(in Chinese)
[29] WANG Q,ZHAO R,CUI Y,et al.Rapid and simple determination of sarafloxacin and difloxacin in beef by capillary electrophoresis coupled with solid-phase extraction[J].Journal of Analytical Methods in Chemistry,2018,2018:1981436.
[30] 李海迪,杨先乐,胡鲲,等.高效液相色谱法测定中华绒螯蟹主要组织中双氟沙星及其代谢产物沙拉沙星方法[J].药物分析杂志,2009,29(7):1142-1147. LI H D,YANG X L,HU K,et al.RP-HPLC determination of difloxacin and its metabolite in tissues of Chinese mitten-handed crab (Eriocheir sinensis)[J].Chinese Journal of Pharmaceutical Analysis,2009,29(7):1142-1147.(in Chinese)

Establishment of an Indirect Competitive Chemiluminescence Enzyme-linked Immunoassay for Sarafloxacin

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