克伦特洛单克隆抗体的研制和初步鉴定
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摘要
本文研究β-兴奋剂类药物克伦特洛(Clenbuterol,CL)的单克隆抗体，为建立克伦特洛残留检测的免疫学方法奠定基础。采用重氮化法将克伦特洛苯环上的氨基重氮化后，与载体蛋白人血清白蛋白(HSA)、牛血清白蛋白(BSA)等偶联。合成的人工抗原CL-HSA、CL-BSA等进行紫外扫描鉴定，结果表明人工抗原偶联成功。选取40只5～7周龄的健康雌性Balb/c小鼠随机分成10组，第1～8组为试验组，以CL-HSA作为免疫原，分为125、100、75、50μg/只四个剂量组，每相邻两组剂量相同，奇数组间隔2周，偶数组间隔3周，第9组和10组分别为佐剂对照组和空白对照组。第二次加强免疫后，用双向免疫琼脂扩散监测抗血清效价及半抗原特异性。高剂量组抗体上升较快，但特异性较差。第四次加强免疫后，高低剂量组及两种间隔方案得到的抗血清差别不明显。取双向免疫琼脂扩散效价达1∶16且特异性较好的小鼠的血清，建立阳性克隆筛选的间接ELISA方法。用方阵滴定法确定间接ELISA最适包被浓度为2μg·ml~(-1)。待免疫小鼠休息1个月以上，选取1只免疫反应强的小鼠，融合前3天加强免疫，取脾细胞1×10~8个与1×10~7个SP2/0骨髓瘤细胞融合。融合后得到的96孔培养板的平均克隆生长率达到39.16％，经间接ELISA筛选，平均阳性率为4.20％。选取阳性最强的三个
华中农业大学2001届硕士学位论文
克隆孔A一76、C一710和C·611进行克隆化。经三次克隆化后，取
得3株杂交瘤细胞株，分别为CLA一716、CLC一710、CLC一611。
体内接种小鼠产生腹水，检测其效价分别为1.6义105、1.24、1护、
9.1、105，用间接抑制ELIsA法检测稀释4万倍腹水的特异性，
产生500，0抑制(xe，。)的eL的浓度分别为一ong劝1.!、l000ng，1.’、
1 oong·ml’，。
木研究表明，用重氮化法合成的人工免疫原(CL一HSA)免
疫原性良好。用CL一HSA致敏的脾淋巴细胞与小鼠SPZ/0骨髓瘤
细胞融合得到三株分泌高效价的克伦特洛特异性的单克隆抗体
的杂交瘤细胞。这些优质的单克隆抗体将在克伦特洛的残留分析
中发挥应有的作用，对于提高国内违禁药物克伦特洛等日一兴奋
剂的分析水平有着很大的实用价值和应用前景。
In order to obtain hybridoma cell line producing monoclonal antibody against clenbuterol (CL), CL was diazotized and directly conjugated with three kinds of carrier protein, Human Sera Albumin (HSA), Bovine Sera Albumin (BSA) and Ovalbumin (OVA). The conjugates were identified by uv-visible recording spectrophotometer, which demonstrate that the artificial antigens were synthesized successfully.
Using CL-HSA as an immunogen, antiserum was taken from Balb/c mice's tail vein and detected by double agarose immunodiffusion (DADT). After the fourth booster injection, the highest titer of antiserum is 1: 16. No significant difference in titer value and in specifity was observed between four doses (125 vs 100vs 75 vs 50ug per mouse) of immunogen and two breaks (2 vs 3 weeks). Two of the mice were scarified and antiserum was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) with 2 g/ml CL-OVA as coating antigen for selecting the positive cloning wells.
Stimulated splenic lymphocytes from the mouse giving best immunizing response were fused with SP2/0 myeloma cells to produce hybridomas. 21 stable positive wells were selected by ELISA and 3 highest positive wells of A-76, C-710 and C-611 were cloning. 3 stable hybrids CLA-76, CLC-710, CLC-611 producing monoclonal antibodies, of some subclass of IgG, were isolated after three cloning. The ELISA tilers of the ascites were 1.6 105, 1.24 l06, and 9.1 10s respectively, and the specificity of the
McAbs is measured by competitive inhibition enzyme linked immunosorbent assay, in which 1: 4 104 of ascites were used. The consentration of clenbuterol inhibiting 50% (ICso) for CLA-76, ILC-710, CLC-611 were 10 ng ml-1, 103 ng ml-11, 102 ng -ml-1. The McAbs specific for clenbuterol will be very useful for studying the immunoassay in the determination of clenbuterol residues in biological sample.
华中农业大学2001届硕士学位论文
克隆孔A一76、C一710和C·611进行克隆化。经三次克隆化后，取
得3株杂交瘤细胞株，分别为CLA一716、CLC一710、CLC一611。
体内接种小鼠产生腹水，检测其效价分别为1.6义105、1.24、1护、
9.1、105，用间接抑制ELIsA法检测稀释4万倍腹水的特异性，
产生500，0抑制(xe，。)的eL的浓度分别为一ong劝1.!、l000ng，1.’、
1 oong·ml’，。
木研究表明，用重氮化法合成的人工免疫原(CL一HSA)免
疫原性良好。用CL一HSA致敏的脾淋巴细胞与小鼠SPZ/0骨髓瘤
细胞融合得到三株分泌高效价的克伦特洛特异性的单克隆抗体
的杂交瘤细胞。这些优质的单克隆抗体将在克伦特洛的残留分析
中发挥应有的作用，对于提高国内违禁药物克伦特洛等日一兴奋
剂的分析水平有着很大的实用价值和应用前景。
In order to obtain hybridoma cell line producing monoclonal antibody against clenbuterol (CL), CL was diazotized and directly conjugated with three kinds of carrier protein, Human Sera Albumin (HSA), Bovine Sera Albumin (BSA) and Ovalbumin (OVA). The conjugates were identified by uv-visible recording spectrophotometer, which demonstrate that the artificial antigens were synthesized successfully.
Using CL-HSA as an immunogen, antiserum was taken from Balb/c mice's tail vein and detected by double agarose immunodiffusion (DADT). After the fourth booster injection, the highest titer of antiserum is 1: 16. No significant difference in titer value and in specifity was observed between four doses (125 vs 100vs 75 vs 50ug per mouse) of immunogen and two breaks (2 vs 3 weeks). Two of the mice were scarified and antiserum was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) with 2 g/ml CL-OVA as coating antigen for selecting the positive cloning wells.
Stimulated splenic lymphocytes from the mouse giving best immunizing response were fused with SP2/0 myeloma cells to produce hybridomas. 21 stable positive wells were selected by ELISA and 3 highest positive wells of A-76, C-710 and C-611 were cloning. 3 stable hybrids CLA-76, CLC-710, CLC-611 producing monoclonal antibodies, of some subclass of IgG, were isolated after three cloning. The ELISA tilers of the ascites were 1.6 105, 1.24 l06, and 9.1 10s respectively, and the specificity of the
McAbs is measured by competitive inhibition enzyme linked immunosorbent assay, in which 1: 4 104 of ascites were used. The consentration of clenbuterol inhibiting 50% (ICso) for CLA-76, ILC-710, CLC-611 were 10 ng ml-1, 103 ng ml-11, 102 ng -ml-1. The McAbs specific for clenbuterol will be very useful for studying the immunoassay in the determination of clenbuterol residues in biological sample.
引文
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2 Rivire JE, Spoo JW Chemical intissues of food animals [A]. ADAMS. Veterinary PHarmacology and therapentic [M] 7th ed. Ames: Iowa State University Bress, 1995, 1148-1156
3 Kuiper HAY, Noordam MMH, Van DF. Illegal uses of β-adrenergic agonists: European Community. Journal of Animal Science, 1998, 76(1) Jan: 195-207
4 陈杖榴，杨桂香，孙永学等．兽药残留毒性与生态毒理学研究进展[J]．华南农业大学学报，2001．22(1)：88-91
5 段九华．动物可食性组织中环丙沙星戏留的ELISA方法研究[J]．华中农业大学硕士论文．武汉，1998
6 庄仁礼，李英超，刘子良等．应加强对动物产品中有害物质残留的监督和检测[J]．辽宁兽牧兽医，1999，6：30-31
7 Smith DJ. The pharmacokinetics, metabolism and tissue residues of β-adrenergists in livestock, Journal of Animal Science, 1998, 76: 173-194
8 Aldo P. Bioanalysis of β_2-agonists by hyphenated Chromatographic and mass spectrometric techniques. Journal of Chromatography B, 1996, 687: 27-42
9 杨藻宸主编．药理学和药物学治疗学[M]．上册．北京：人民出版社，第一版，426-427
10 Locer JW, Joseph MG, Kathleen PH, et al. Determination of Clenbuterol in cattle, Sheep, and Swine Tissues by electron Ionization Gas Chromatography Mass Spectrometry. Journal of AOAC Chemists International, 1994, 77(4): 917-924
11 Baker PK, Dalrymple RH, Ingle DL et al. Use of a β-adrenergic antagonist to alter muscle and fat deposition in lambs. Journal of Animal
Science, 1984, 59: 1256
12 Dalrymple RH, Baker RK, Gingher DE, et al. A repartitioning agent to improve performance and carcass composition of broilers. Poultry Science, 1984, 63: 2376
13 Yang YT and Eceugolt MA. Multiple action of adrenergic agonists on skeletal and aclipose Tissue. Journal of Biochemistry, 1986, 261: 1-10
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