【作者】 杨正涛；

【导师】 张乃生；

【作者基本信息】 中国人民解放军军需大学 ， 临床兽医学， 2003， 硕士

【摘要】 克伦特罗（Clenbuterol，CL）是一种常被非法用作促生长添加剂的β₂-受体激动剂，彻底查禁CL的非法滥用，必须要有高效、灵敏、特异的残留检测方法。本研究利用偶氮化法制备出CL完全抗原，用杂交瘤技术制备出CL单克隆抗体，建立了CL固相抗体ELISA残留检测方法，试验结果如下： 在阴暗避光4℃的条件下，用亚硝酸钠使CL发生偶氮化反应，偶氮化CL与载体蛋白酪氨酸残基上的酚羟基反应，从而生成“CL-载体蛋白”偶联物。分别制备出CL-KLH、CL-BSA和CL-OVA三种偶联物，CL-KLH与CL-BSA分别免疫小鼠制备抗血清，用CL-OVA包被酶标板，检测到的抗体可以和游离的CL发生特异性的结合反应。 用CL-KLH免疫BALB／c小鼠，连续5次，间隔3周。最后一次免疫后的第3d，无菌采取脾脏，脾细胞用聚乙二醇与SP2／O骨髓瘤细胞融合，用96孔细胞培养板，HAT 1640选择性培养基，在37℃，5％CO₂条件下半量换液培养。第11d换液时对有细胞克隆生长的培养孔，以CL-OVA包被酶标板，间接ELISA检测阳性孔，阳性孔有限稀释法连续3次克隆，最后获得了四株稳定分泌CL-McAb的杂交瘤细胞株，分别是1D6、1H5、1H7和2F10，四株杂交瘤培养上清液效价分别达到1：1100、1：1200、1：900和1：700。腹水效价都在6×10⁵以上，对CL亲和力从强至弱依次为1D6＞1H5＞1H7＞2F10。对沙丁胺醇交叉反应性测定，结果最小的是1D6，只有2.32％的交叉反应件。1H5、1H7和2F10交叉反应性依次为12.9％、26％和＜5％。抗原识别位点分析结果表明，这四株McAb至少识别两个不同的抗原位点。 用辣根过氧化物酶标记CL，得到了酶标抗原HRP-CL，用CL单克隆抗体包被酶标反应板，10％兔血清封闭，侍检测样品中的CL与HRP-CL竞争CL-McAb上的结合位点，建立起固相抗体竞争ELISA，CL-McAb最佳包被浓度为1.25μg／ml，酶标抗原的最佳稀释倍数为1：400，最适检测范围为1～100ng／ml，从而建立了快速检测CL的固相抗体竞争ELISA方法。更多还原

【Abstract】 This paper reported the preparation of CL-McAb and the development of enzyme immunoassay for the detection of clenbuterol residues.β2- agonist clenbuterol has been used as bronchiodilators in human medicine for over 30 years. Unfortunately, this agent has also been used illegally now as growth-promoter in livestock production. Its illegal use will led to toxic effects after human consumption of meat products. The analytical method for clenbuterol residues detection is urgently needed.Clenbuterol was a hapten. To prepare antigen, clenbuterol was conjugated to carrier proteins KLH, BSA and OVA by diazotization. CL-KLH and CL-BSA were used for the immunization of mice and CL-OVA was used as the coating antigen for the ELISA study. Both antiserums of CL-KLH and CL-BSA could bind specifically with CL. The anti-clenbuterol antibody titer of antiserum immunized with CL-KLH was higher than that with CL-BSA.Fore hybridoma secreting monoclonal antibodies against CL were achieved by injection of BALB/c mice with CL-KLH. These four McAbs were characterized for the cross reactivities, specificities, neutralizing abilities vs. CL and relative affinity. The titer degree of ascites fluid of these four McAb was 1 : 3×106~1 : 6×105. 1D6 showed 3.98% cross reaction with Salbutamol. Relative affinity showed 1D6>1H5>1H7>2F10.Clenbuterol was diazotized to be labelled with HRP. HRP-CL could bind with CL-McAb specifically. The samples was added in the ELISA plat wellswhich were coated with CL-McAb(lD6) and blocked with 10% rabbit serum. CL in the samples could bind with CL-McAb. After washing, the HRP-CL was added and incubated for 15min at 37℃. After the plat was washed again, OPD-H2O2 was added and the reaction was terminated by adding 2M H2SO4 in 15 min. The OD490nm values was read at 490nm in a microplat reader. The contraction of CL-McAb and dilution of HRP-CL were determined. The standard curve was obtained. This immunoassay provided a analytical method for the detection of clenbuterol residues and could be use in preparation of clenbuterol ELISA kit.更多还原