检测猪尿、猪肉中莱克多巴胺的化学发光间接竞争免疫分析方法的建立  

Development of an indirect competitive chemiluminescence immunoassay for determining ractopamine residues in swine meat and urine

作　　者：王权[1] 赵新宇[2] 蒋蔚[1] 陈永军[1] 刘迎春[1] 宋宁宁[1] 石金磊[1] 

机构地区：[1]中国农业科学院上海兽医研究所/农业部寄生虫学重点开放实验室,上海200241 [2]上海出入境检验检疫局,上海201300

出　　处：《畜牧与兽医》2013年第3期17-21,共5页Animal Husbandry & Veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费专项资金项目(2010JB06)

摘　　要：将莱克多巴胺(RAC)与戊二酸酐反应,用混合酸酐法使反应产物与牛血清白蛋白(BSA)偶联生成免疫原,并采用淋巴细胞杂交瘤技术制备RAC单克隆抗体,建立检测猪尿、猪肉中莱克多巴胺化学发光间接竞争免疫分析法。结果显示:飞行质谱法测得RAC-戊二酸单酯分子量为416.2,RAC-戊二酸单酯与BSA偶联比为3.4∶1;RAC单克隆抗体最佳稀释倍数为1∶8×104,包被抗原最佳质量浓度为0.5μg/mL;标准曲线呈线性,回归方程为Y=-0.188 2x+0.392 2,R2=0.993 4,IC50值为0.267 ng/mL;与RAC类似化合物及其他药品无明显的交叉反应。尿样回收率为76.8%～90.8%,批内变异系数为6.8%～11.5%,批间变异系数为7.2%～13.8%;猪肉样回收率为75.2%～87.4%,批内变异系数为7.1%～11.6%,批间变异系数为8.6%～14.1%。结果表明:用RAC-戊二酸单脂与BSA偶联物作免疫原成功地研制了针对RAC的特异性抗体,以单抗为基础建立了检测猪尿、猪肉中莱克多巴胺化学发光间接竞争免疫分析法,为进一步研制快速筛选检测试剂盒奠定了基础。The experiment was conducted to establish an indirect competitive chemiluminescence immunoassay for determining ractopamine （RAC） residues based on monoclonal antibody （McAb）. Ractopamine was coupled to bovine serum albumin （BSA） by mixture anhydrides method. The RAC-BSA conjugate was used as immunogen and McAb was prepared using lymphocytes hybridoma technology. The indirect competitive chemiluminescence immunoassay based on McAb was developed for detection and estimation of RAC residues in swine meat and urine. The molecular weight of RAC-glutaricacid mono-lipid was 416. 2 by MALDI TOF-MS/MS. The couple ratio of RAC-BSA was about 3.4 ： 1. The optimal concertration of the coating antigen （RAC-OVA） and that of McAb against RAC were 0. 5 μg/mL and 1 ： 80 000, respectively. Linear detection of standard curve covered a range from 0. 01 ng/mL to 100ng/mL （ Y=-0. 188 2x＋0. 392 2, R2 = 0. 993 4）. There was no significant cross-reactivity with those compounds similar to RAC and other drugs. The fortified recovery of swine urine was between 76. 8% and 90. 8%, and the intra-assay and inter-assay coefficients of variation ranged from 6. 8% to 11.5% and from 7.2% to 13. 8%, respectively. The fortified recovery of swine meat was between 75.2% and 87.4%, and the intra-assay and inter-assay coefficients of variation were from 7. 1% to 11.6% and from 8.6% to 14. 1%, respectively. The results indicated that the anti-RAC antibodies were successfully prepared using RAC-BSA conjugate as immunogen An indirect competitive chemiluminescence immunoassay for detecting racto- pamine residues in swine meat and urine was developed based on McAb. This study will apply an importannt basis for the development of rapid test kits for screening ractopamine.

关 键 词：莱克多巴胺 单克隆抗体 化学发光间接竞争免疫分析法 

分 类 号：S859.84[农业科学—临床兽医学]