莱克多巴胺单克隆抗体免疫检测试剂的研制和应用
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摘要
莱克多巴胺(Ractopamine,RCT)是一种β肾上腺素能兴奋剂,具有营养再分配、促进蛋白质合成的作用。莱克多巴胺可能被非法作为动物饲料添加剂,以提高饲料转化率和增加瘦肉率,但其残留对人类健康存在潜在的危害。本研究旨在制备特异性针对莱克多巴胺的单克隆抗体,并建立竞争酶联免疫吸附试验(ciELISA)和免疫胶体金试纸条用于莱克多巴胺残留的检测。
采用混合酸酐法将莱克多巴胺与匙孔蓝蛋白(KLH)、牛血清白蛋白(BSA)偶联,经紫外光谱扫描确定偶联成功,测得偶联物RCT-KLH浓度为3.6mg/mL,RCT-BSA浓度为6.2mg/mL.以偶联物RCT-KLH作为免疫原,免疫6～8周龄雌性BALB/c小鼠,取小鼠脾细胞和骨髓瘤细胞SP2/0-Ag-14进行融合。以RCT-BSA作为包被抗原,经间接ELISA筛选出阳性细胞株,再用有限稀释法进行多次亚克隆,获得稳定分泌单克隆抗体的杂交瘤细胞10株,分别为1D9、1F3、2C11、5C3、3A10、4A8、687、6D5、7C11、7D3,其中单克隆抗体5C3和3A10间接ELISA效价均为1：500000。间接竞争ELISA显示,单克隆抗体5C3对莱克多巴胺的50%阻断作用浓度(IC50)为3.98ng/mL,对盐酸克伦特罗和沙丁胺醇的IC50均大于2000ng/mL；对盐酸克伦特罗和沙丁胺醇的交叉反应率均小于0.2%。
以单克隆抗体5C3为核心试剂,建立了间接竞争ELISA检测莱克多巴胺的方法,并研制出ELISA试剂盒。以混合酸酐法人工合成的莱克多巴胺-卵清蛋白(RCT-OVA)为包被抗原,莱克多巴胺为竞争的半抗原,两者与一定量的抗莱克多巴胺单克隆抗体5C3在竞争体系中反应。试验结果表明,包被抗原的最佳稀释浓度为1：1600,抗莱克多巴胺单克隆抗体5C3工作浓度为1：8000,包被抗原37℃包被3h为最佳包被条件。间接竞争ELISA检测莱克多巴胺标准品检测线性范围为0.5～100ng/mL,最低检测限为0.5ng/mL,标准曲线的相关系数(R2)达到0.9968。试剂盒的样品检测离散系数(CV)不高于3.4%,阳性添加回收率平均达到99.2%。对猪饲料、肝脏、尿液样品,ELISA试剂盒的检测限分别为0.01mg/kg.0.01mg/kg和0.5ng/mL。经厦门出入境检验检疫局检验检疫技术中心、四川出入境检验检疫局、南通市畜产品质量检验测试中心和南京市栖霞区畜牧兽医站等四家单位对人工添加莱克多巴胺样品进行检测,结果显示当样品中莱克多巴胺浓度>0.5ng/mL(?)寸,本研究中研制的ELISA检测试剂盒与德国拜法食品安全检测和饲料监控分析公司生产的莱克多巴胺ELISA检测试剂盒的检测符合率达95%以上。
采用混合酸酐法制备的RCT-OVA偶联抗原和胶体金标记莱克多巴胺单克隆抗体5C3,建立了检测莱克多巴胺的胶体金免疫层析试验方法,并组装成标准化检测卡。该检测卡具有较好的敏感性和特异性,最低可检测1Ong/mL的莱克多巴胺,而与克伦特罗和沙丁胺醇无交叉反应。应用该方法对饲料、组织和尿液样品中莱克多巴胺的最低检测量分别为0.05mg/kg、0.05mg/kg和10ng/mL.经厦门出入境检验检疫局技术中心、四川出入境检验检疫局、南通市畜产品质量检验测试中心和南京市栖霞区畜牧兽医站等四家单位对人工添加样品进行检测,结果显示当样品中莱克多巴胺残留浓度>10ng/mL时,本研究中研制的胶体金免疫检测试纸条与德国拜法食品安全检测和饲料监控分析公司生产的莱克多巴胺ELISA检测试剂盒检测结果符合率均为100%；当样品中莱克多巴胺残留浓度>5ng/ml时,符合率可达85%以上。
应用莱克多巴胺单克隆抗体免疫检测试剂检测了不同来源的样品。在江苏检验检疫局检测猪肉样品655份,莱克多巴胺ELISA试剂盒与LC-MSMS法对猪肉样品检测的符合率为100%。在吉林检验检疫局检测出口猪副产品31批次,莱克多巴胺未检出。国家质量安全监控计划部分2008年8-10月份检测进口冻猪副产品751批和盐溃肠衣69批,莱克多巴胺不合格率6.95%；2009年8-10月检测进口猪副产品869批,莱克多巴胺不合格率1.57%。
Ractopamine is one of the β-adrenergic agonists, which can redistribute nutrition, promote protein synthesis, enhance lean meat to fat ration and improve feed conversion efficiency. However, accumulative residue of ractopamine in food producing animals can cause symptoms of food poisoning in human. Considering that the illegal use of ractopamine in livestock production would be harmful for human after consumption of meat products containing residual ractopamine, ractopamine was limited or banned to be utilized as growth promoters in livestock breeding. Our research aimed to prepare monoclonal antibodies against ractopamine for establishment the cELISA method and colloidal gold strips for detection the residual ractopamine.
In this study ractopamine was conjugated with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) based on mannish reaction, and the synthesized antigens were identified by ultraviolet radiation, with the concentrations of3.6mg/ml and6.2mg/ml respectively. The spleen cells of BALB/c mice immunized by RCT-KLH were fused with SP2/0-Ag-14myeloma cells using PEG4000. The RCT-BSA was used for the coating antigen for indirect enzyme linked immunosorbent assay (ELISA) to screen hybridoma cells, and limited dilution method was performed to subclone the positive clones. Nearly10cell lines including5C3and3A10, which stably secreted monoclonal antibodies against ractopamine, were obtained. The indirect ELISA titers of ascites fluids of5C3and3A10were1:5x105. In addition, competitive indirect ELISA (ciELISA) was established based on the monoclonal antibodies, and the IC50detected with ciELISA was3.98ng/ml for ractopamine, whereas the McAb had no cross-reactivity with either clenbuterol or salbutamol.
Next, an indirect competitive ELISA kit was developed using the monoclonal antibody5C3and manipulation of the kit was investigated thoroughly. The optimal condition for the kit was as following:Coating at37℃for3h, then blocking another3h at37℃with1% gelatin, besides, the optimal concentration for coated antigen was1:1600, while the optimal working dilution titer of McAb was1:8000. The linear range of ciELISA to detect ractopamine was0.5ng/ml～100ng/ml and minimum detectable concentration was0.5ng/ml. The minimal amount of ractopamine to be detected from samples of pig fodder, liver, urine were0.01mg/kg,0.01mg/kg and0.5ng/ml, respectively.
We further developed a colloidal gold strip to detect ractopamine using ractopamine-OVA conjugated antigen and monoclonal antibody specific to ractopamine labeled with colloidal gold. Results indicated that the strip has high sensitivity and specificity with detecting limitation of10ng/mL of ractopamine and no cross-reactivity to clenbuterol and salbutamol. The minimal amount of ractopamine to be detected from samples of pig fodder, tissue and urine were0.05mg/kg,0.05mg/kg and lOng/ml, respectively.
In summary, we developed immunoassay kits including an ELISA kit and colloidal gold strips to detect residual ractopamine and the kits have been used in clinical detection by some govern departments. Clinical results indicated that the kits may replace the use of similar kits from foreign companies and have a promising future in application.
采用混合酸酐法将莱克多巴胺与匙孔蓝蛋白(KLH)、牛血清白蛋白(BSA)偶联,经紫外光谱扫描确定偶联成功,测得偶联物RCT-KLH浓度为3.6mg/mL,RCT-BSA浓度为6.2mg/mL.以偶联物RCT-KLH作为免疫原,免疫6～8周龄雌性BALB/c小鼠,取小鼠脾细胞和骨髓瘤细胞SP2/0-Ag-14进行融合。以RCT-BSA作为包被抗原,经间接ELISA筛选出阳性细胞株,再用有限稀释法进行多次亚克隆,获得稳定分泌单克隆抗体的杂交瘤细胞10株,分别为1D9、1F3、2C11、5C3、3A10、4A8、687、6D5、7C11、7D3,其中单克隆抗体5C3和3A10间接ELISA效价均为1：500000。间接竞争ELISA显示,单克隆抗体5C3对莱克多巴胺的50%阻断作用浓度(IC50)为3.98ng/mL,对盐酸克伦特罗和沙丁胺醇的IC50均大于2000ng/mL；对盐酸克伦特罗和沙丁胺醇的交叉反应率均小于0.2%。
以单克隆抗体5C3为核心试剂,建立了间接竞争ELISA检测莱克多巴胺的方法,并研制出ELISA试剂盒。以混合酸酐法人工合成的莱克多巴胺-卵清蛋白(RCT-OVA)为包被抗原,莱克多巴胺为竞争的半抗原,两者与一定量的抗莱克多巴胺单克隆抗体5C3在竞争体系中反应。试验结果表明,包被抗原的最佳稀释浓度为1：1600,抗莱克多巴胺单克隆抗体5C3工作浓度为1：8000,包被抗原37℃包被3h为最佳包被条件。间接竞争ELISA检测莱克多巴胺标准品检测线性范围为0.5～100ng/mL,最低检测限为0.5ng/mL,标准曲线的相关系数(R2)达到0.9968。试剂盒的样品检测离散系数(CV)不高于3.4%,阳性添加回收率平均达到99.2%。对猪饲料、肝脏、尿液样品,ELISA试剂盒的检测限分别为0.01mg/kg.0.01mg/kg和0.5ng/mL。经厦门出入境检验检疫局检验检疫技术中心、四川出入境检验检疫局、南通市畜产品质量检验测试中心和南京市栖霞区畜牧兽医站等四家单位对人工添加莱克多巴胺样品进行检测,结果显示当样品中莱克多巴胺浓度>0.5ng/mL(?)寸,本研究中研制的ELISA检测试剂盒与德国拜法食品安全检测和饲料监控分析公司生产的莱克多巴胺ELISA检测试剂盒的检测符合率达95%以上。
采用混合酸酐法制备的RCT-OVA偶联抗原和胶体金标记莱克多巴胺单克隆抗体5C3,建立了检测莱克多巴胺的胶体金免疫层析试验方法,并组装成标准化检测卡。该检测卡具有较好的敏感性和特异性,最低可检测1Ong/mL的莱克多巴胺,而与克伦特罗和沙丁胺醇无交叉反应。应用该方法对饲料、组织和尿液样品中莱克多巴胺的最低检测量分别为0.05mg/kg、0.05mg/kg和10ng/mL.经厦门出入境检验检疫局技术中心、四川出入境检验检疫局、南通市畜产品质量检验测试中心和南京市栖霞区畜牧兽医站等四家单位对人工添加样品进行检测,结果显示当样品中莱克多巴胺残留浓度>10ng/mL时,本研究中研制的胶体金免疫检测试纸条与德国拜法食品安全检测和饲料监控分析公司生产的莱克多巴胺ELISA检测试剂盒检测结果符合率均为100%；当样品中莱克多巴胺残留浓度>5ng/ml时,符合率可达85%以上。
应用莱克多巴胺单克隆抗体免疫检测试剂检测了不同来源的样品。在江苏检验检疫局检测猪肉样品655份,莱克多巴胺ELISA试剂盒与LC-MSMS法对猪肉样品检测的符合率为100%。在吉林检验检疫局检测出口猪副产品31批次,莱克多巴胺未检出。国家质量安全监控计划部分2008年8-10月份检测进口冻猪副产品751批和盐溃肠衣69批,莱克多巴胺不合格率6.95%；2009年8-10月检测进口猪副产品869批,莱克多巴胺不合格率1.57%。
Ractopamine is one of the β-adrenergic agonists, which can redistribute nutrition, promote protein synthesis, enhance lean meat to fat ration and improve feed conversion efficiency. However, accumulative residue of ractopamine in food producing animals can cause symptoms of food poisoning in human. Considering that the illegal use of ractopamine in livestock production would be harmful for human after consumption of meat products containing residual ractopamine, ractopamine was limited or banned to be utilized as growth promoters in livestock breeding. Our research aimed to prepare monoclonal antibodies against ractopamine for establishment the cELISA method and colloidal gold strips for detection the residual ractopamine.
In this study ractopamine was conjugated with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) based on mannish reaction, and the synthesized antigens were identified by ultraviolet radiation, with the concentrations of3.6mg/ml and6.2mg/ml respectively. The spleen cells of BALB/c mice immunized by RCT-KLH were fused with SP2/0-Ag-14myeloma cells using PEG4000. The RCT-BSA was used for the coating antigen for indirect enzyme linked immunosorbent assay (ELISA) to screen hybridoma cells, and limited dilution method was performed to subclone the positive clones. Nearly10cell lines including5C3and3A10, which stably secreted monoclonal antibodies against ractopamine, were obtained. The indirect ELISA titers of ascites fluids of5C3and3A10were1:5x105. In addition, competitive indirect ELISA (ciELISA) was established based on the monoclonal antibodies, and the IC50detected with ciELISA was3.98ng/ml for ractopamine, whereas the McAb had no cross-reactivity with either clenbuterol or salbutamol.
Next, an indirect competitive ELISA kit was developed using the monoclonal antibody5C3and manipulation of the kit was investigated thoroughly. The optimal condition for the kit was as following:Coating at37℃for3h, then blocking another3h at37℃with1% gelatin, besides, the optimal concentration for coated antigen was1:1600, while the optimal working dilution titer of McAb was1:8000. The linear range of ciELISA to detect ractopamine was0.5ng/ml～100ng/ml and minimum detectable concentration was0.5ng/ml. The minimal amount of ractopamine to be detected from samples of pig fodder, liver, urine were0.01mg/kg,0.01mg/kg and0.5ng/ml, respectively.
We further developed a colloidal gold strip to detect ractopamine using ractopamine-OVA conjugated antigen and monoclonal antibody specific to ractopamine labeled with colloidal gold. Results indicated that the strip has high sensitivity and specificity with detecting limitation of10ng/mL of ractopamine and no cross-reactivity to clenbuterol and salbutamol. The minimal amount of ractopamine to be detected from samples of pig fodder, tissue and urine were0.05mg/kg,0.05mg/kg and lOng/ml, respectively.
In summary, we developed immunoassay kits including an ELISA kit and colloidal gold strips to detect residual ractopamine and the kits have been used in clinical detection by some govern departments. Clinical results indicated that the kits may replace the use of similar kits from foreign companies and have a promising future in application.
引文
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36. Rodemann H P. The stimulation of protein degradation in muscle by Ca2+ismediated by PGE and does not require the calcium2 activated protease [J]. J Biol Chem,1982,257:1632-1638.
37. Mills S E, Liu C Y. Sensitivity of lipolysis and lipogenesis to dibutyry 12 cAMPand-adrenergic agonists in swine adipocytes in vitro [J]. J Anim Sci 1990,68:1024-1029.
38. Grant A L, Sklearlund D M, Helferich W G, et al. Skeletal muscle growth and expression of skeletal mucle alpha-actin mRNA and insulin-like growth fector I mRNA in pigs during feeding and withdrawal of ractopamine[J]. J Anim Sci,1993,71:19-26.
39. Smith DJ, Feil V J,Huwe J K, et al.Metabolism and disposition of ractopamine hydrochloride by turkey poults [J]. Drug Metabolism and Disposition,1993,21:624-633.
40. Smith DJ, Giddings J M, Feil V J, et al. Identification o f ractopamine hydrochloride metabolites excreted into rat bile [J]. Xenobiotic,1995,25:511-520.
41. Huston H, Hawkins D R, Paulson GD, et al. Xenobiotics and food producing animals: metabolismand residues[M]. USA:American Chemical Society,1992:234-243.
42. Werner T L. Muscle protein metabolism in finishing pigs fed ractopamine [J]. J Anim Sci 1989,67: 2255-2262.
43. Crome P K, Mckeith F K, Carr T R, et al. Effect of ractopamine on growth performance, carcass composition, and cutting yields of pigs slaughtered at 107 and 125 kilograms [J]. J Anim Sci,1996,74: 709-716.
44. Peterla TA, Scanes C G. Effect of beta-adrenergic agonists on lipolysis and lipogenesis by porcine adipose tissue in vitro [J]. J Anim Sci,1990,68:1024-1029.
45. Smith D J, Paulson GD. Growth characteristics of rats receiving ractopamine hydrochloride and the metabolic disposition of ractopamine hydrochloride after oral or intraperitioneal adminstrati on [J]. J AnimSci,1994,72:404-414.
46. Jean P A,Philippe M, Abarouno L B. et al. Identification of ractopamine residues in tissue and urine samples ultratrace level us ing liquid chromatography-positive electrospray tandem mass spectrometry[J].Journal o f chromatography B,2002,774:59-66.
47. Martinez Navarro J F. Food poisoning related to consumption of illicit beta-agonist in liver [J]. Lancet,1990,336:1311.
48.蒋志伟,卜仕金,张雨梅,等.莱克多巴胺对鼠的毒性研究[J].兽药与饲料添加剂,2001(6)：4-6.
49. Lommen A, Haasnoot W, Weseman J M. Nuclear Magnetic resonance controlled method for coupling of fenoterol to a carrier and enzyme[J]. Food and Agriculture Immunology,1995,7:123-129
50.张海棠,王自良,王艳荣,等.莱克多巴胺的毒性及其残留免疫学检测技术研究进展[J].安徽农业科学,2006,34(18)：4534-4536
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52.于洪侠,杨曙明.莱克多巴胺残留检测方法研究进展[J].中国兽药杂志,2004,38(11)：44-47.
53. TurbergM P, Rodewald JM, ColemanM R. Determination of ractopamine hydrochloride in monkey plasma and swine serum by high performance liquid chromatography with electrochemical detection [J]. J Chromatogr B Biomed,1996,675:279-285.
54. TurbergM P, Macy T D, Lewis J J, et al. Determination of ractopamine hydrochloride in swine, cattle, and turkey feeds by liquid chromatography with coulometric detection [J]. J AOAC Int,1994,77: 840-847.
55. TurbergM P, Macy T D, Lewis J J, et al. Determination of ractopamine hydrochloride in swine and turkey tissues by liquid chromatography with coulometric detection [J]. J AOAC Int,1995,78: 1394-1402.
56. David JB, Barbara N, Joe M. Radioimmunoassay of imipramine and desmethylimipramine[J]. Life Sciences,1978,1 (22):137-146
57. DicksonLC, MacNeil J D, LeeS, etal. Determination of beta-agonist residues in bovine urine using liquid chromatography-tandem mass spectrometry[J]. J AOAC Int,2005,88 (1):46-56
58. Blanca J, Munoz P,Morgado M, et al. Determination of clenbuterol, ractopamine and zilpaterol in liver and urine by liquid chromatography tandem mass spectrometry [J]. Analytica ChimicaActa,2005, 529:199-205
59. Kaita De Wach. Hubert De Brabander and Dirk Courtheyn. LC-MS/MS to detect and identify four beta agonists and quantify clenbuterol in liver[J]. Analyst,1998,123:2701-2705
60. Shelver W L, Smith DJ. Immunoaffinity column as sample cleanup method for determination of the beta-adrenergic agonist ractopamine and its metabolites[J]. J. AOAC Int.2002,85 (6):1302-1307
61. Zhang L Y, Chang B Y, Dong T.et al. Simultaneous determination of salbutamol, ractopamine, and clenbuterol in animal feeds by SPE and LC-MS. J Chromatogr Sci 2009,47(4):324-328.
62. Inoue T., Chang J P. Capillary electrophoretic separation and quantitation of ractopamine stereoisomers using cyclodextrins as chiral additives[J]. Journal of Liquid Chromatography and Related Technologies,2003,26(11):1677-1693
63. Shelver WL., Smith DJ. Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis:Comparative study with HPLC and ELISA[J]. Journal of Agricultural and Food Chemistry,2003,51 (13):3715-3721
64. Shelver W L, Smith D J, Berry E S. Production and characterization of a monoclonal antibody against the β-adrenergic agonist ractopamine [J]. Journal of Agricultural and Food Chemistry,2000,48:4020-4026
65. Shelver W L, Smith D J. Application of a monoclonal antibody based enzyme linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals [J]. Journal of Agricultural and Food Chemistry,2002,50(10):2742-2747
66. Wicker A L, Turberg M P, Coleman M R. Evaluation of ractopamine cross-reactivity in several commercially available (3-agonist enzyme immunoassay kits[J]. Analyst,1995,120:2879-2881.
67. Haasnoot W, Stouten P, Lommen A, et al. Determination of fenoterol and ractopamine in urine by enzyme immunoassay [J]. Analyst,1994,119:2675-2680.
68. Elliott C T, Thompson C S, Arts C J M, et al. Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the-agonist [J]. Analyst,1998, 123:1103-1107.
69. ShelverW L, Smith D J. Development of an immunoassay for the-adrenergic agonist ractopamine [J]. J Immunoassay,2000,21:1223.
70. Shelver WL, Smith DJ. Development of an immunoassay for the-adrenergic agonist ractopamine[J]. Journal of Immunoassay,2000,21 (1):1-23
71. Zhang M Z, Wang M Z, Chen Z L, et al. Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine [J]. Anal Bioanal Chem.2009,1618-2650 (Electronic)
[1]Smith DJ, Ehrenfried KM, Dalidowicz JD, etal. Binding of ractopamine HCl to ocular tissues of cattle and turkeys in vivo and to melanin in vitro[J]. J. Anim. Sci.,2002b, 80:2931-2941.
[2]Turberg MP, Macy TD, Lewis JJ, et al. Determination of ractopamine hydrochloride in swine、 cattle, and turkey feeds by liquid chromatography with coulometric detection[J]. J. AOAC Int,1994, July 1,77(4):840-847.
[3]HaasnootW, Stouten P, LommenA, Determination of fenoterol and ractopamine in urine by enzyme immunoassay [J]. Analyst,1994,119(12):2675-2680
[4]Shelver WL and Smith DJ. Development of an immunoassay for the β-adrenergic agonist ractopamine [J]. J Immunoassay,2000,21(1):1-23.
[5]于洪侠,杨曙明,朱宇.莱克多巴胺多克隆抗体的制备.中国兽医科学[J],2006,36(1)：62-65
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[2]Turberg MP, Macy TD, Lewis JJ, etal. Determination of ractopamine hydrochloride in swine^ cattle, and turkey feeds by liquid chromatography with coulometric detection[J]. J AOAC Int, 1994, July 1,77(4):840-847.
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[2]Turberg MP, Macy TD, Lewis JJ, etal. Determination of ractopamine hydrochloride in swine, cattle and turkey feeds by liquid chromatography with coulometric detection[J]. J AOAC Int,1994, July 1,77 (4):840-847.
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32. Eshaq(Isaac) Shishani, Sin Chii,Sami Jamokha. Determination of ractopamine in animal tissues by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry[J].Analytica Chimica Acta,2003,483:137-145
33. Smith D J. The pharmacokinetics, metabolism and tissue residues of β-adrenergic agonosts in livestock [J]. J Anim Sci,1998,76:173-194.
34. Yang Y T, Mcelligott M A. Multiple action of β-adrenergic agonist on skeletal muscle and adipose tissue [J]. Biochem J,1989,261:1210.
35. Helferich W G. Skeletal musclea-actin synthesis is increased protein translationally in pigs fed phenethanolamine ractopamine [J]. Endocrinology,1990,126:3096-3190.
36. Rodemann H P. The stimulation of protein degradation in muscle by Ca2+ismediated by PGE and does not require the calcium2 activated protease [J]. J Biol Chem,1982,257:1632-1638.
37. Mills S E, Liu C Y. Sensitivity of lipolysis and lipogenesis to dibutyry 12 cAMPand-adrenergic agonists in swine adipocytes in vitro [J]. J Anim Sci 1990,68:1024-1029.
38. Grant A L, Sklearlund D M, Helferich W G, et al. Skeletal muscle growth and expression of skeletal mucle alpha-actin mRNA and insulin-like growth fector I mRNA in pigs during feeding and withdrawal of ractopamine[J]. J Anim Sci,1993,71:19-26.
39. Smith DJ, Feil V J,Huwe J K, et al.Metabolism and disposition of ractopamine hydrochloride by turkey poults [J]. Drug Metabolism and Disposition,1993,21:624-633.
40. Smith DJ, Giddings J M, Feil V J, et al. Identification o f ractopamine hydrochloride metabolites excreted into rat bile [J]. Xenobiotic,1995,25:511-520.
41. Huston H, Hawkins D R, Paulson GD, et al. Xenobiotics and food producing animals: metabolismand residues[M]. USA:American Chemical Society,1992:234-243.
42. Werner T L. Muscle protein metabolism in finishing pigs fed ractopamine [J]. J Anim Sci 1989,67: 2255-2262.
43. Crome P K, Mckeith F K, Carr T R, et al. Effect of ractopamine on growth performance, carcass composition, and cutting yields of pigs slaughtered at 107 and 125 kilograms [J]. J Anim Sci,1996,74: 709-716.
44. Peterla TA, Scanes C G. Effect of beta-adrenergic agonists on lipolysis and lipogenesis by porcine adipose tissue in vitro [J]. J Anim Sci,1990,68:1024-1029.
45. Smith D J, Paulson GD. Growth characteristics of rats receiving ractopamine hydrochloride and the metabolic disposition of ractopamine hydrochloride after oral or intraperitioneal adminstrati on [J]. J AnimSci,1994,72:404-414.
46. Jean P A,Philippe M, Abarouno L B. et al. Identification of ractopamine residues in tissue and urine samples ultratrace level us ing liquid chromatography-positive electrospray tandem mass spectrometry[J].Journal o f chromatography B,2002,774:59-66.
47. Martinez Navarro J F. Food poisoning related to consumption of illicit beta-agonist in liver [J]. Lancet,1990,336:1311.
48.蒋志伟,卜仕金,张雨梅,等.莱克多巴胺对鼠的毒性研究[J].兽药与饲料添加剂,2001(6)：4-6.
49. Lommen A, Haasnoot W, Weseman J M. Nuclear Magnetic resonance controlled method for coupling of fenoterol to a carrier and enzyme[J]. Food and Agriculture Immunology,1995,7:123-129
50.张海棠,王自良,王艳荣,等.莱克多巴胺的毒性及其残留免疫学检测技术研究进展[J].安徽农业科学,2006,34(18)：4534-4536
51.张中亚,徐海聂,瞿进文.D=兴奋剂的监测方法概述[J].肉品卫生,2000,187(1)：8-12
52.于洪侠,杨曙明.莱克多巴胺残留检测方法研究进展[J].中国兽药杂志,2004,38(11)：44-47.
53. TurbergM P, Rodewald JM, ColemanM R. Determination of ractopamine hydrochloride in monkey plasma and swine serum by high performance liquid chromatography with electrochemical detection [J]. J Chromatogr B Biomed,1996,675:279-285.
54. TurbergM P, Macy T D, Lewis J J, et al. Determination of ractopamine hydrochloride in swine, cattle, and turkey feeds by liquid chromatography with coulometric detection [J]. J AOAC Int,1994,77: 840-847.
55. TurbergM P, Macy T D, Lewis J J, et al. Determination of ractopamine hydrochloride in swine and turkey tissues by liquid chromatography with coulometric detection [J]. J AOAC Int,1995,78: 1394-1402.
56. David JB, Barbara N, Joe M. Radioimmunoassay of imipramine and desmethylimipramine[J]. Life Sciences,1978,1 (22):137-146
57. DicksonLC, MacNeil J D, LeeS, etal. Determination of beta-agonist residues in bovine urine using liquid chromatography-tandem mass spectrometry[J]. J AOAC Int,2005,88 (1):46-56
58. Blanca J, Munoz P,Morgado M, et al. Determination of clenbuterol, ractopamine and zilpaterol in liver and urine by liquid chromatography tandem mass spectrometry [J]. Analytica ChimicaActa,2005, 529:199-205
59. Kaita De Wach. Hubert De Brabander and Dirk Courtheyn. LC-MS/MS to detect and identify four beta agonists and quantify clenbuterol in liver[J]. Analyst,1998,123:2701-2705
60. Shelver W L, Smith DJ. Immunoaffinity column as sample cleanup method for determination of the beta-adrenergic agonist ractopamine and its metabolites[J]. J. AOAC Int.2002,85 (6):1302-1307
61. Zhang L Y, Chang B Y, Dong T.et al. Simultaneous determination of salbutamol, ractopamine, and clenbuterol in animal feeds by SPE and LC-MS. J Chromatogr Sci 2009,47(4):324-328.
62. Inoue T., Chang J P. Capillary electrophoretic separation and quantitation of ractopamine stereoisomers using cyclodextrins as chiral additives[J]. Journal of Liquid Chromatography and Related Technologies,2003,26(11):1677-1693
63. Shelver WL., Smith DJ. Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis:Comparative study with HPLC and ELISA[J]. Journal of Agricultural and Food Chemistry,2003,51 (13):3715-3721
64. Shelver W L, Smith D J, Berry E S. Production and characterization of a monoclonal antibody against the β-adrenergic agonist ractopamine [J]. Journal of Agricultural and Food Chemistry,2000,48:4020-4026
65. Shelver W L, Smith D J. Application of a monoclonal antibody based enzyme linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals [J]. Journal of Agricultural and Food Chemistry,2002,50(10):2742-2747
66. Wicker A L, Turberg M P, Coleman M R. Evaluation of ractopamine cross-reactivity in several commercially available (3-agonist enzyme immunoassay kits[J]. Analyst,1995,120:2879-2881.
67. Haasnoot W, Stouten P, Lommen A, et al. Determination of fenoterol and ractopamine in urine by enzyme immunoassay [J]. Analyst,1994,119:2675-2680.
68. Elliott C T, Thompson C S, Arts C J M, et al. Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the-agonist [J]. Analyst,1998, 123:1103-1107.
69. ShelverW L, Smith D J. Development of an immunoassay for the-adrenergic agonist ractopamine [J]. J Immunoassay,2000,21:1223.
70. Shelver WL, Smith DJ. Development of an immunoassay for the-adrenergic agonist ractopamine[J]. Journal of Immunoassay,2000,21 (1):1-23
71. Zhang M Z, Wang M Z, Chen Z L, et al. Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine [J]. Anal Bioanal Chem.2009,1618-2650 (Electronic)
[1]Smith DJ, Ehrenfried KM, Dalidowicz JD, etal. Binding of ractopamine HCl to ocular tissues of cattle and turkeys in vivo and to melanin in vitro[J]. J. Anim. Sci.,2002b, 80:2931-2941.
[2]Turberg MP, Macy TD, Lewis JJ, et al. Determination of ractopamine hydrochloride in swine、 cattle, and turkey feeds by liquid chromatography with coulometric detection[J]. J. AOAC Int,1994, July 1,77(4):840-847.
[3]HaasnootW, Stouten P, LommenA, Determination of fenoterol and ractopamine in urine by enzyme immunoassay [J]. Analyst,1994,119(12):2675-2680
[4]Shelver WL and Smith DJ. Development of an immunoassay for the β-adrenergic agonist ractopamine [J]. J Immunoassay,2000,21(1):1-23.
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[6]曲勃,齐孟文.莱克多巴胺单克隆抗体的制备与初步鉴定.核农学报[J],2005,19(5)：393-396
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