氯唑西林可视化免疫亲和凝胶柱检测方法研究
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摘要
为了建立氯唑西林(Cloxacillin,CLOX)可视化免疫亲和凝胶柱检测方法。实验采用活化酯法,将辣根过氧化物酶(HRP)与氯唑西林偶联,制得氯唑西林酶标抗原,并通过紫外光谱扫描鉴定表明CLOX酶标抗原合成成功。实验采用恒温振荡法,将溴化氰活化的琼脂糖凝胶分别与氯唑西林抗体、HRP抗体偶联制得氯唑西林抗体胶和HRP抗体胶,作为亲和凝胶柱的检测层与质控层。建立氯唑西林可视化免疫亲和凝胶柱分析方法,确定了该方法在酶标抗原的稀释倍数为4000、抗体胶稀释倍数为10、HRP胶稀释倍数为20、样品稀释液pH值为5.7、加样时间为60 s条件下,达到最佳工作状态。该方法检测限为25μg/L,特异性强,与哌拉西林、阿莫西林、氨苄青霉素、四环素、呋喃它酮、甲砜霉素、链霉素均无交叉反应,动物源食品实际样品中氯唑西林检出限为25μg/kg。该方法便捷、快速、可视化,适合作为高通量筛选氯唑西林的有效检测手段。
In order to establish a method for the detection of cloxacillin visualized immunoaffinity gel column, the activated ester method was used to combine horseradish peroxidase(HRP) with cloxacillin to obtain the cloxacillin enzyme standard antigen. Results of the UV spectrum scanning showed that the CLOX enzyme standard antigen was successfully synthesized. The agarose gel activated by hydrogen bromide was combined with cloxacillin antibody and HRP antibody, respectively, which was used as the detection layer and the quality control layer of the affinity gel column, respectively. A visual immunoaffinity gel column analysis method for cloxacillin was established. The optimum condition of this method was the dilution ratio of enzyme-labeled antigen of 4000, dilution ratio of antibody gel of 10, dilution ratio of HRP gel of 20, sample dilution pH of 5.7 and sample addition time of 60 s. The detection limit of this method was 25 μg/L. Its specificity was strong.There was no cross-reaction with piperacillin, amoxicillin, ampicillin, tetracycline, furantanone, thiamphenicol and streptomycin. The detection limit of cloxacillin in animal-derived foods was 25 μg/kg. This method is convenient, rapid and visual. It is suitable for high-throughput screening of cloxacillin.
In order to establish a method for the detection of cloxacillin visualized immunoaffinity gel column, the activated ester method was used to combine horseradish peroxidase(HRP) with cloxacillin to obtain the cloxacillin enzyme standard antigen. Results of the UV spectrum scanning showed that the CLOX enzyme standard antigen was successfully synthesized. The agarose gel activated by hydrogen bromide was combined with cloxacillin antibody and HRP antibody, respectively, which was used as the detection layer and the quality control layer of the affinity gel column, respectively. A visual immunoaffinity gel column analysis method for cloxacillin was established. The optimum condition of this method was the dilution ratio of enzyme-labeled antigen of 4000, dilution ratio of antibody gel of 10, dilution ratio of HRP gel of 20, sample dilution pH of 5.7 and sample addition time of 60 s. The detection limit of this method was 25 μg/L. Its specificity was strong.There was no cross-reaction with piperacillin, amoxicillin, ampicillin, tetracycline, furantanone, thiamphenicol and streptomycin. The detection limit of cloxacillin in animal-derived foods was 25 μg/kg. This method is convenient, rapid and visual. It is suitable for high-throughput screening of cloxacillin.
引文
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[16]章敏,黄显会,杨刚,等.牛奶中氯唑西林残留量的高效液相色谱法测定[J].动物医学进展,2012,33(6):56-60ZHANG Min,HUANG Xian-hui,YANG Gang,et al.Determination of cloxacillin in residues in milk by high-performance liquid chromatography[J].Progress in Veterinary Medicine,2012,33(6):56-60
[2]CHEN X X,GAN M,HONG X F,et al.Development of a rapid and sensitive quantum dot-based immunochromatographic strip by double labeling PCRproducts for detection of Staphylococcus aureus in food[J].Food Control,2014,46(12):225-232
[3]LUO J P,JIANG W X,Beier Ross C,et al.Development of an enzyme-linked immunosorbent assay for determination of the furaltadone etabolite,3-Amino-5-Morpholinomethyl-2-Oxazolidinone(AMOZ)in animal tissues[J].Biomedical and Environmental Sciences,2012,25(4):449-457
[4]HE J,WU N,LUO P J,et al.Development of a heterologous enzyme-linked immunosorbent assay for the detection of clindamycin and lincomycin residues in edible animal tissues[J].Meat Science,2017,125(3):137-142
[5]FENG B,DAI Y Z,WANG L,et al.A novel affinity ligand for polystyrene surface from a phage display random library and its application in anti-HIV-1 ELISA system[J].Biologicals,2008,37(1):48-54
[6]田园.展青霉素特异性抗体的制备及其免疫学检测方法的研究[D].泰安:山东农业大学,2012TIAN Yuan.Preparation of patulin specific antibodies and their application in development of immunoassay for patulin[D].Tai’an:Shandong Agricultural University,2012
[7]刘宣兵,张改平,侯玉泽,等.庆大霉素人工抗原的合成与鉴定[J].黑龙江畜牧兽医,2008,12:64-65LIU Xuan-bing,ZHANG Gai-ping,HOU Yu-ze,et al.Synthesis and identification of the antigen for gentamicin[J].Heilongjiang Animal Science and Veterinary Medicine,2008,12:64-65
[8]闫叶娜.呋喃西林代谢物单克隆抗体的制备及其免疫分析方法的建立[D].南京:南京农业大学,2010YAN Ye-na.Preparation of monoclonal antibody for nitrofurazone and development of its immunity analysis[D].Nanjing:Nanjing Agricultural University,2010
[9]周克楠,唐勇,蓝彩凤,等.呋喃唑酮代谢物单克隆抗体及其新型免疫层析检测试纸条的制备[J].中国生物制品学杂志,2014,27(7):927-931ZHOU Ke-nan,TANG Yong,LAN Cai-feng,et al.Preparation of monoclonal antibody against furazolidone metabolite and novel immunochromatographic strips[J].Chinese Journal of Biologicals,2014,27(7):927-931
[10]柳爱春,刘超,赵芸,等.免疫胶体金法快速检测水产品中硝基呋喃类代谢物的研究[J].浙江农业学报,2013,25(1):95-102LIU Ai-chun,LIU Chao,ZHAO Yun,et al.Rapid determination of nitrofuran metabolites in aquatic products by colloidal gold method[J].Acta Agriculture Zhejiangensis,2013,25(1):95-102
[11]吴巧丽,叶春生.胶体金免疫层析技术快速检测沙丁胺醇残留[J].现代食品科技,2012,11:1595-1599WU Qiao-li,YE Chun-sheng.Colloidal gold immunochromatographic assay for residual sabutamo[J].Modern Food Science and Technology,2012,11:1595-1599
[12]吕月霞,王瑞,黄登宇,等.呋喃它酮代谢物直接竞争化学发光酶免疫分析法的建立[J].食品工业科技,2015,36(22):71-75,86LYU Yue-xia,WANG Rui,HUANG Deng-yu,et al.Development of a direct competitive chemiluminescent enzyme immunoassay for detection of furaltadone metabolite[J].Science and Technology of Food Industry,2015,36(22):71-75,86
[13]TIAN Xi,DONG Yang-qing,WANG Yun-feng,et al.Quantification of diethyl phthalate by a rapid and homogenous fluroescene polarization immunoassay[J].Analytical Letters,2015,48(18):2843-2855
[14]Mc Cracken R J,Van Rhijn J A,Kennedy D G.Transfer of nitrofuran residues from parent broiler breeder chickens to broiler progeny[J].British Poultry Science,2005,46(3):287-292
[15]杨典原,吕月霞,李亚楠,等.化学发光酶免疫法检测呋喃它酮代谢物残留[J].畜牧与兽医2016,48(4):112-115YANG Dian-yuan,LYU Yue-xia,LI Ya-nan,et al.Competitive chemiluminescent enzyme immunoassay for detection of furaltadone metabolite[J].Animal Husbandry&Veterinary Medicine,2016,48(4):112-115
[16]章敏,黄显会,杨刚,等.牛奶中氯唑西林残留量的高效液相色谱法测定[J].动物医学进展,2012,33(6):56-60ZHANG Min,HUANG Xian-hui,YANG Gang,et al.Determination of cloxacillin in residues in milk by high-performance liquid chromatography[J].Progress in Veterinary Medicine,2012,33(6):56-60