高亲和力克伦特罗单克隆抗体的研制及免疫试纸检测方法的建立  

Generation of High Affinity Monoclonal Antibody and Development of Colloidal Gold-Labeled Strip for Rapid Detection of Clenbuterol

作　　者：张海棠[1] 王自良[1] 刘玺[2] 钟华[1] 范国英[1] 

机构地区：[1]河南科技学院动物科学学院,新乡453003 [2]河南科技学院食品学院,新乡453003

出　　处：《生物技术通报》2008年第5期149-153,共5页Biotechnology Bulletin

基　　金："十一五"国家科技支撑计划重大项目"农药;兽药抗体库建立"资助(2006BAK02A21/1)

摘　　要：重氮化法将克伦特罗(CL)偶联于BSA合成免疫抗原BSA-CL,戊二醛法(GA)合成包被抗原OVA-CL,用BSA-CL免疫Balb/C小鼠。细胞融合技术建立高亲和力CL单克隆抗体(CLmAb)杂交瘤细胞株,体内诱生腹水法制备CLmAb,并测定其亲和性。依据胶体金免疫层析试验(GICA)原理,应用CLmAb研制CL残留快速检测免疫试纸(CL-Strip),并测定其性能。结果表明,筛选出16株杂交瘤细胞,其中亲和力最高的为3C12-6H6,亲和常数(Ka)为1.76×1010L/mol。CL-Strip的标准曲线呈典型的S型,符合4参数logit曲线拟合,目测检测限为0.5μg/L;其敏感性与竞争ELISA试剂盒和GC/MS相当,符合率为100%。CL-Strip具有具有敏感、特异、快速、简便的特点,可推广应用于CL残留的快速检测。The diazotization method was used to conjugate clenbuterol （CL）to bovine serum albumin（BSA）to prepare artificial immunogen BSA-CL,and the glutaraldehyde（GA）method was used to couple CL to ovalbumin（OVA）to prepare coating antigen OVA-CL. Balb/C mice were immunized with BSA-CL. Hybridoma lines that secrete high affinity monoclonal antibody against CL（CL mAb）,were filtered with cell fusion and the affinity of CL mAb were characterized. Base on the principle of gold immunochromatography assay （GICA）,a stript for detection CL （CL-Strip）was developed and its traits were tested. The results showed that sixteen hybridoma lines were filtered and the best one was 3C12- 6H6,which has a high attinity constant （Ka）with 1.76 × 10^10 L/mol. The calibration curve of CL-Strip,as typical sigmoid curve,fitted to the four parameter logistic,with detection limit 0.5μg/L and the sensitivity as same as that of ELISA-Kit and GC/MS,the coincidence rate was 100% compared with ELISA-Kit and GC/MS. The CL-Strip possessed rapidity, sensitivity,specificity and briefness,thus it was proved to be used for the rapid test of CL residues in animal food.

关 键 词：克伦特罗 细胞融合 单克隆抗体 胶体金免疫层析试验 快速检测试纸 

分 类 号：S859.84[农业科学—临床兽医学] Q813[农业科学—兽医学]