苯乙醇胺A单克隆抗体的研制及ELISA检测方法的建立
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摘要
为制备苯乙醇胺A(phenylethanolamine A,PA)单克隆抗体,建立一种针对苯乙醇胺A的快速、简便、灵敏度高的检测方法,本试验采用重氮化法将制备的苯乙醇胺A衍生物分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)进行偶联作为免疫原和包被原。利用弗氏佐剂充分乳化免疫原(PA-BSA),按常规免疫程序免疫6～8周龄的雌性BALB/c小鼠,选择血清抗体效价较高的小鼠,取其脾细胞与SP2/0骨髓瘤细胞以PEG法进行细胞融合。结果显示,经3次细胞亚克隆筛选后,最终筛选出一株可稳定分泌抗苯乙醇胺A单克隆抗体的杂交瘤细胞株,命名为D6H8,利用此细胞以小鼠体内诱生法制备抗体。经鉴定,D6H8腹水抗体亚型为IgG1,轻链为κ链;纯化后的抗体与沙丁胺醇、盐酸克伦特罗、盐酸异丙肾上腺素和盐酸去氧肾上腺素均无明显的交叉反应(CR<0.18%),表明该抗体特异性良好。利用此抗体建立针对苯乙醇胺A药物残留检测的间接竞争ELISA方法,结果表明,腹水抗体效价为1∶12 800,猪肉中苯乙醇胺A添加浓度在5～1 000 ng/mL时线性关系良好,标准工作曲线为y=0.3861x-0.1845 (R~2=0.990),其IC_(50)为58.88 ng/mL,LOD为3.83 ng/mL,回收率在85.96%～104.32%之间。综上所述,本试验成功建立了检测苯乙醇胺A药物残留的间接竞争ELISA方法,该方法灵敏度高、稳定性较好。
To prepare monoclonal antibodies against phenylethanolamine A(PA) and establish a fast,simple and sensitive method for the detection of PA,a derivative of PA was coupled with bovine serum albumin(BSA) and ovalbumin(OVA) as immunogen and coating antigen by diazotization.The immunogen that emulsified by Freund's adjuvant was used for immunization of 6 to 8 weeks' BALB/c mice in accordance with conventional procedures.The immune spleen cells of mice with high serum antibody titer were fused with SP2/0 myeloma cells by PEG method.After three subclonal screening,a hybridoma cell strain D6 H8 of secreting anti-PA monoclonal antibody was screened out and the inducing ascites in vivo method was employed to produce monoclonal antibodies.The McAb was identified to be IgG1 subtype and kappa light chain.The purified antibody showed no cross-reactions with salbutamol amine,clenbuterol hydrochloride,isoprenaline hydrochloride and deoxyepinephrine hydrochloride(CR<0.18%),indicating that the antibody had good specificity.An indirect competitive ELISA method for PA drug residues was established using this antibody.The results showed that the titer of ascites antibody was 1∶12 800 and the linear relationship was good when the concentration of PA in pork was 5 to 1 000 ng/mL.The linear equation was y=0.3861 x-0.1845(R~2=0.990).The concentration of IC_(50)and LOD were 58.88 and 3.83 ng/mL,respectively.The recovery rates were between 85.96% and 104.32%,indicating that the ELISA method was reproducible and stable.In summary,an indirect competitive ELISA method for the determination of PA residues had been successfully established with high sensitivity and good stability.
To prepare monoclonal antibodies against phenylethanolamine A(PA) and establish a fast,simple and sensitive method for the detection of PA,a derivative of PA was coupled with bovine serum albumin(BSA) and ovalbumin(OVA) as immunogen and coating antigen by diazotization.The immunogen that emulsified by Freund's adjuvant was used for immunization of 6 to 8 weeks' BALB/c mice in accordance with conventional procedures.The immune spleen cells of mice with high serum antibody titer were fused with SP2/0 myeloma cells by PEG method.After three subclonal screening,a hybridoma cell strain D6 H8 of secreting anti-PA monoclonal antibody was screened out and the inducing ascites in vivo method was employed to produce monoclonal antibodies.The McAb was identified to be IgG1 subtype and kappa light chain.The purified antibody showed no cross-reactions with salbutamol amine,clenbuterol hydrochloride,isoprenaline hydrochloride and deoxyepinephrine hydrochloride(CR<0.18%),indicating that the antibody had good specificity.An indirect competitive ELISA method for PA drug residues was established using this antibody.The results showed that the titer of ascites antibody was 1∶12 800 and the linear relationship was good when the concentration of PA in pork was 5 to 1 000 ng/mL.The linear equation was y=0.3861 x-0.1845(R~2=0.990).The concentration of IC_(50)and LOD were 58.88 and 3.83 ng/mL,respectively.The recovery rates were between 85.96% and 104.32%,indicating that the ELISA method was reproducible and stable.In summary,an indirect competitive ELISA method for the determination of PA residues had been successfully established with high sensitivity and good stability.
引文
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[17] LI X,WANG W,WANG L,et al.Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay[J].Analytical & Bioanalytical Chemistry,2015,407(25):7615-7624.
[18] MI J著.董晓云译.自动在线固相萃取-液相色谱-串联质谱法对动物源性食品中3种β受体激动剂的快速检测[J].中国畜牧兽医,2014,41(7):125. MI J(Author).DONG X Y(Translator).Rapid detection of three β-agonists in animal-derived foods by automatic on-line solid phase extraction-liquid chromatography-tandem mass spectrometry[J].China Animal Husbandry & Veterinary Medicine,2014,41(7):125.(in Chinese)
[19] 郑百芹,李爱军,张建民,等.液相色谱-串联质谱法测定猪肉中3种β-受体激动剂类药物残留[J].中国畜牧兽医,2012,39(3):224-227. ZHENG B Q,LI A J,ZHANG J M,et al.Determination of 3 β-agonists in pork by liquid chromatography-tandem mass spectrometry[J].China Animal Husbandry & Veterinary Medicine,2012,39(3):224-227.(in Chinese)
[20] 李丹妮,黄华,张婧,等.3种β-受体激动剂荧光免疫层析试纸条的制备及特性研究[J].中国畜牧兽医,2017,44(8):2437-2442. LI D N,HUANG H,ZHANG J,et al.Preparation and properties analysis of three β2-agonists fluorescent immunochromatographic strip[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2437-2442.(in Chinese)
[21] 周炜,朱聪英,应永飞,等.高效液相色谱-串联质谱法测定动物尿液中23种β-受体激动剂[J].中国兽药杂志,2013,47(9):46-51. ZHOU W,ZHU C Y,YING Y F,et al.Determination of 23 β-agonists in animal urine by high performance liquid chromatography-tandem mass spectrometry[J].Chinese Journal of Veterinary Drug,2013,47(9):46-51.(in Chinese)
[22] 焦奎,张书圣.酶联免疫分析技术及应用[M].北京:化学工业出版社,2004. JIAO K,ZHANG S S.Enzyme-linked Immunosorbent Assay and Its Application[M].Beijing:Chemical Industry Press,2004.(in Chinese)
[23] 宋娟,王榕妹,王悦秋,等.半抗原的设计、修饰及人工抗原的制备[J].分析化学,2010,38(8):1211-1218. SONG J,WANG R M,WANG Y Q,et al.Hapten design,modification and preparation of artificial antigens[J].Chinese Journal of Analytical Chemistry,2010,38(8):1211-1218.(in Chinese)
[24] HERMANSON G T.Bioconjugate Techniques[M].London:Academic Press,2008.
[2] COURTHEYN D,BIZEC B L,BRAMBILLA G,et al.Recent developments in the use and abuse of growth promoters[J].Analytica Chimica Acta,2002,473(1):71-82.
[3] SCRAMLIN S M,PLATTER W J,GOMEZ R A,et al.Comparative effects of ractopamine hydrochloride and zilpaterol hydrochloride on growth performance,carcass traits,and longissimus tenderness of finishing steers[J].Journal of Animal Science,2010,88(5):1823-1829.
[4] ENGESETH N J,LEE K O,BERGEN W G,et al.Fatty acid profiles of lipid depots and cholesterol concentration in muscle tissue of finishing pigs fed ractopamine[J].Journal of Food Science,2010,57(5):1060-1062.
[5] BRAMBILLA G,CENCI T,FRANCONI F,et al.Clinical and pharmacological profile in a clenbuterol epidemic poisoning of contaminated beef meat in Italy[J].Toxicology Letters,2000,114(1-3):47-53.
[6] MEI L,CAO B,YANG H,et al.Development of an immunoaffinity chromatography column for selective extraction of a new agonist phenylethylamine A from feed,meat and liver samples[J]. Journal of Chromatography B,2014,945-946:178-184.
[7] 中国人民共和国农业部公告第1519号[J].饲料研究,2011,5:30. Announcement No.1519th of the Ministry of Agriculture of the People’s Republic of China[J].Feed Research,2011,5:30.(in Chinese)
[8] 顾亮,丁磊.液相色谱串联质谱法测定饲料中盐酸克伦特罗、莱克多巴胺、沙丁胺醇、苯乙醇胺A[J].化学分析计量,2012,21(1):37-39. GU L,DING L.Determination of clenbuterol,ractopamine,salbuterol and nylethanolamine in feeds by liquid chromatography-electrospray tandem mass spectrometry[J].Chemical Analysis and Meterage,2012,21(1):37-39.(in Chinese)
[9] COURANT F,PINEL G,BICHON E,et al.Development of a metabolomic approach based on liquid chromatography-high resolution mass spectrometry to screen for clenbuterol abuse in calves[J].Analyst,2009,134(8):1637-1646.
[10] YANG H J.Simultaneous determination of 10 β2-agonists in swine urine using liquid chromatography-tandem mass spectrometry and multi-walled carbon nanotubes as a reversed dispersive solid phase extraction sorbent[J].Journal of Chromatography A,2012,1260:25-32.
[11] CABAN M,STEPNOWSKI P,KWIATKOWSKI M,et al.Determination of β-blockers and β-agonists using gas chromatography and gas chromatography-mass spectrometry——A comparative study of the derivatization step[J].Journal of Chromatography A,2011,1218(44):8110-8122.
[12] 樊宇飞,苏晓鸥,刘萌.超高效液相色谱测定猪配合饲料中苯乙醇胺A的研究[J].中国畜牧兽医,2013,40(5):225-228. FAN Y F,SU X O,LIU M.Determination of phenylethanolamine A in formula feed by using ultra performance liquid chromatography[J].China Animal Husbandry & Veterinary Medicine,2013,40(5):225-228.(in Chinese)
[13] BOZZOLINO C,LEPORATI M,GANI F,et al.Development and validation of an UHPLC-MS/MS method for β2-agonists quantification in human urine and application to clinical samples[J].Journal of Pharmaceutical & Biomedical Analysis,2017,150:15-24.
[14] MI Q,WANG Z W,CHAI C Y,et al.Multilayer structured immunosensor based on a glassy carbon electrode modified with multi-wall carbon nanotubes,polythionine,and gold nanoparticles[J].Microchimica Acta,2011,173(3-4):459-467.
[15] 刘佳,张凯,龙瑞军,等.不同β-受体激动剂添加模式对肉牛血液生化指标和激素水平的影响[J].中国畜牧兽医,2017,44(9):2638-2643. LIU J,ZHANG K,LONG R J,et al.Different feeding mode of β-agonists treatments on plasma biochemical parameters and internal hormone levels in cattle[J].China Animal Husbandry & Veterinary Medicine,2017,44(9):2638-2643.(in Chinese)
[16] CAO B,HE G,YANG H,et al.Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS)[J].Talanta,2013,115(17):624-630.
[17] LI X,WANG W,WANG L,et al.Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay[J].Analytical & Bioanalytical Chemistry,2015,407(25):7615-7624.
[18] MI J著.董晓云译.自动在线固相萃取-液相色谱-串联质谱法对动物源性食品中3种β受体激动剂的快速检测[J].中国畜牧兽医,2014,41(7):125. MI J(Author).DONG X Y(Translator).Rapid detection of three β-agonists in animal-derived foods by automatic on-line solid phase extraction-liquid chromatography-tandem mass spectrometry[J].China Animal Husbandry & Veterinary Medicine,2014,41(7):125.(in Chinese)
[19] 郑百芹,李爱军,张建民,等.液相色谱-串联质谱法测定猪肉中3种β-受体激动剂类药物残留[J].中国畜牧兽医,2012,39(3):224-227. ZHENG B Q,LI A J,ZHANG J M,et al.Determination of 3 β-agonists in pork by liquid chromatography-tandem mass spectrometry[J].China Animal Husbandry & Veterinary Medicine,2012,39(3):224-227.(in Chinese)
[20] 李丹妮,黄华,张婧,等.3种β-受体激动剂荧光免疫层析试纸条的制备及特性研究[J].中国畜牧兽医,2017,44(8):2437-2442. LI D N,HUANG H,ZHANG J,et al.Preparation and properties analysis of three β2-agonists fluorescent immunochromatographic strip[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2437-2442.(in Chinese)
[21] 周炜,朱聪英,应永飞,等.高效液相色谱-串联质谱法测定动物尿液中23种β-受体激动剂[J].中国兽药杂志,2013,47(9):46-51. ZHOU W,ZHU C Y,YING Y F,et al.Determination of 23 β-agonists in animal urine by high performance liquid chromatography-tandem mass spectrometry[J].Chinese Journal of Veterinary Drug,2013,47(9):46-51.(in Chinese)
[22] 焦奎,张书圣.酶联免疫分析技术及应用[M].北京:化学工业出版社,2004. JIAO K,ZHANG S S.Enzyme-linked Immunosorbent Assay and Its Application[M].Beijing:Chemical Industry Press,2004.(in Chinese)
[23] 宋娟,王榕妹,王悦秋,等.半抗原的设计、修饰及人工抗原的制备[J].分析化学,2010,38(8):1211-1218. SONG J,WANG R M,WANG Y Q,et al.Hapten design,modification and preparation of artificial antigens[J].Chinese Journal of Analytical Chemistry,2010,38(8):1211-1218.(in Chinese)
[24] HERMANSON G T.Bioconjugate Techniques[M].London:Academic Press,2008.