苯乙醇胺A单克隆抗体的研制及ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2019.03.025

Abstract

Abstract:

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 D6H8 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=0.3861x-0.1845 (R²=0.990).The concentration of IC₅₀and 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.

Key words: phenylethanolamine A(PA); hybridoma; monoclonal antibody; indirect competitive ELISA; recovery test

CLC Number:

S852.4

XIANG Zilai, WANG Ling, XIA Sugan, ZOU Hui, GU Jianhong, YUAN Yan, LIU Xuezhong, LIU Zongping, BIAN Jianchun. Preparation of Monoclonal Antibodies Against Phenylethanolamine A and Establishment of ELISA Method[J]. , 2019, 46(3): 849-857.

References

[1] 王培龙.β-受体激动剂及其检测技术研究[J].农产品质量与安全,2014,1:44-52. WANG P L.Study on β-receptor agonist and its detection technology[J].Quality and Safety of Agro-Products,2014,1:44-52.(in Chinese)
[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 10β2-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 3β-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 23β-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.

Preparation of Monoclonal Antibodies Against Phenylethanolamine A and Establishment of ELISA Method

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	HOU Haoyu, ZHENG Nannan, WU Hongju, CHEN Yinlong, LI Chao, ZHANG Angke, JI Pengchao, WAN Bo, DU Yongkun, ZHANG Gaiping. Preparation and Characterization of Monoclonal Antibody Against p30 Protein of African Swine Fever Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1241-1249.
[2]	WANG Leming, WANG Yurui, ZENG Zixuan, RAO Guoshun, WU Zhengjiao, JIN Weikun, WANG Dongying. Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 677-686.
[3]	MO Hongfang, JIE Kai, CHEN Xin, WEN Wei, LIU Wenqiang, CHEN Huanchun, QIAN Ping, LI Xiangmin. Preparation and Identification of Monoclonal Antibodies Against Porcine Seneca Valley Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 307-317.
[4]	HU Huanyi, WANG Jianzhong, LIU Changjin, LIN Min, LIU Xiaolan, WEI Huangsiwu, DENG Shunzhou. Eukaryotic Expression of Green Fluorescent Protein and Preparation of Its Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 328-337.
[5]	ZENG Jianyu, DONG Zhenyuan, JIA Wenfeng, ZHANG Guozhong, XUE Jia. Preparation and Identification of Monoclonal Antibody Against Newcastle Disease Virus M Protein [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3423-3431.
[6]	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.
[7]	YANG Songxin, FENG Jianyuan, GUO Xuan, WEI Liuyuan, LIANG Yiying, YAN Hao, ZHANG Ziyi, CHEN Hailan. Preparation of Monoclonal Antibodies for Small Molecular Compounds and Their Applications in the Field of Veterinary Medicine [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 1121-1131.
[8]	LI Jiesen, SUN Ronghang, KUANG Yanqi, CHEN Luman, LIU Qing, GUO Xiaofeng. Optimal Expression of Porcine Epidemic Diarrhea Virus S1D Protein and Preparation of Its Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4641-4651.
[9]	WANG Zhiping, ZHANG Yunjing, SUN Yujie, XU Xin, WANG Zhiyan, HUANG Baicheng, TIAN Kegong. Development and Evaluation of an Indirect ELISA for Detection of Porcine Epidemic Diarrhea Virus IgA in Pig Serum and Milk Captured by Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2248-2255.
[10]	SHI Jianzhou, LI Qingmei, WANG Yanhong, LIU Xiao, LI Ge, GUO Junqing, DENG Ruiguang, ZHANG Gaiping. Establishment of a Method of IPMA for Detection of the Monoclonal Antibody Against H1N1 Swine Influenza Virus [J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2691-2698.
[11]	ZHOU Manli, ZHOU Yucheng, ZHANG Haiwei, QIAO Lianjiang, YANG Yanling. Preparation and Detection of Monoclonal Antibodies Against Mycobacterium bovis MPB70 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(10): 3075-3083.
[12]	FAN Suju, YANG Xingdong, GAO Run. Preparation of Chloramphenicol Monoclonal Antibody and Establishment of ELISA Detection Method [J]. , 2018, 45(9): 2566-2574.
[13]	SONG Hongxin, HAN Bo, LI Shan, XUE Haiyan. Comparison Between Indirect Competitive ELISA and Indirect ELISA in Detecting Bovine κ-casein [J]. , 2018, 45(4): 1114-1119.
[14]	WANG Lingling, SUN Guopeng, XU Ruonan, HE Haixun, YAN Zhanpeng, WANG Yaping, XING Ruilin, REN Pengju, LI Peng, ZHANG Yanfang, ZHU Yanping, YUE Feng, WANG Xuannian. Study on Preparation and Biological Function of Chicken PD-1 Monoclonal Antibody [J]. , 2018, 45(12): 3363-3370.
[15]	JIA Qi-zhen, CHEN Gen-yuan, WANG Lian-qun, YASEN·Maimaiti, WANG Shuai. Preparation and Identification of Monoclonal Antibody Against Swainsonine [J]. , 2017, 44(7): 2155-2159.