环丙沙星单克隆抗体的制备及其免疫学特性分析  

Generation of the monoclonal antibody against Ciprofloxacin and analysis for its immunological traits

作　　者：胡鲲[1] 黄宣运[1] 姜有声[1] 方伟[2] 杨先乐[1] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]广东省水生动物疫病预防控制中心,广州510220

出　　处：《中国免疫学杂志》2010年第6期538-543,共6页Chinese Journal of Immunology

基　　金：上海市科技兴农重点攻关项目(6660106477);公益性行业(农业)科研专项(nyhyzx07-042);公益性行业(农业)科研专项(200803013);现代农业产业技术体系建设专项资金(NYCYTX-49-17)资助

摘　　要：目的:制备环丙沙星单克隆抗体,并分析其免疫学特性,为建立环丙沙星残留的免疫学检测方法服务。方法:用人工抗原环丙沙星-小牛血清白蛋白偶联物免疫BALB/c小鼠,产生预期免疫应答后,将小鼠脾细胞与瘤细胞融合,经初筛、复筛和再克隆,筛选得到1C9、3F6、6H2、6A7、6G11和8F56株分泌环丙沙星单克隆抗体的杂交瘤细胞。对单克隆抗体的亚型、纯度、亲和力、灵敏度及特异性等免疫学特性进行鉴定和分析。结果:1C9、3F6和6A7的抗体亚型为IgG2a;6H2和8F5的抗体亚型为IgG1;6G11的抗体亚型为IgG3。SDS-PAGE电泳结果显示单抗蛋白重链分子量约为50kD,轻链分子量约为25kD。6株细胞产生的单抗均具有良好的特异性和灵敏度。其中,细胞株1C9细胞培养上清和腹水效价分别为1∶6.4×102和1∶5.6×105,亲和力常数可达2.85×109L/mol,IC50值可达245.86ng/ml,最低检测限为45.25ng/ml,对氧氟沙星、双氟沙星、沙拉沙星、左氧氟沙星、孔雀石绿、氯霉素、呋喃西林等药物几乎不存在交叉反应,与恩诺沙星存在交叉反应,交叉反应率为84.6%。结论:本方法制备的环丙沙星单克隆抗体具有较好的亲和力和特异性,可用于环丙沙星残留免疫学检测。Objective：To generate monoclonal antibodies against ciprofloxacin （CIP） and to analyze its immunological traits for establishing a determination method for ciprofloxacin residues in food tissues.Methods：BALB/c mice were immunized by the conjugation of CIP-BSA successfully.The splenic cells of BALB/c mice and the oncocyte were fused and screened in HAT culture medium.Cell strains of 1C9,3F6,6H2,6A7,6G11 and 8F5 were cloned,which could secret the monoclonal antibody （Mab） for ciprofloxacin.Results：By ELISA method,the haplotype of Mabs were identified.The antibody secreted by 1C9,3F6 and 6A7 were classified to the IgG2a.Those of 6H2 and 8F5 were classified to the IgG1 while 6G11 to IgG3.The results of SDS-PAGE showed that the Mab protein was made up of the weight chain and the light chain whose molecular weight were 50 kD and 25 kD,respectively.All of those Mabs had fine specificity and sensibility.The indirect ELISA titer of 1C9 was 1：6.4×102 in supernatant and 1：5.6×105 in ascites.The affinity of 1C9 was 2.85×109 L/mol and the value of IC50 reached to 245.86 ng/ml.The protein secreted by 1C9 was screened for establishing the ELISA method and the detective limitation reached to 45.25 ng/ml.There were no cross reactions to Ofloxacin,Difloxacin,Sarafloxacin,Malachite green,Chloramphenicol and Furacilin while the rate of cross reaction to Enrofloxacin could reach to 84.6%.Conclusion：The Mabs are sensitive and specific,which are suitable to be applied in establishing immunoassay of CIP residues.

关 键 词：环丙沙星 单克隆抗体 亚型 亲和力 交叉反应 

分 类 号：R446.6[医药卫生—诊断学]