莱克多巴胺单克隆抗体的研制及阻断ELISA检测方法的建立  

GENERATION OF MONOCLONAL ANTIBODY AND DEVELOPMENT OF BLOCKING ELISA KIT FOR RACTOPAMINE

作　　者：张海棠[1] 王自良[1] 邓瑞广[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,河南新乡453003 [2]河南省动物免疫学重点实验室,河南郑州450002

出　　处：《核农学报》2008年第6期904-908,共5页Journal of Nuclear Agricultural Sciences

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：用混合酸酐法将莱克多巴胺（RAC）偶联于牛血清白蛋白（BSA）,合成免疫原BSA-RAC,并用紫外和凝胶电泳鉴定;用BSA-RAC免疫Balb/C小鼠,细胞融合技术建立抗RAC单克隆抗体（mAb）杂交瘤细胞株,体内诱生腹水法制备RAC mAb。应用RAC mAb研制RAC残留快速检测阻断ELISA试剂盒（RAC-Kit）,并测定其性能。结果表明,BSA-RAC偶联成功,分子结合比为1∶24.5;筛选出3株杂交瘤细胞,其中最好的4D8株的亲和常数Ka为1.65×1010L/mol。RAC-Kit的标准曲线呈典型的S型,符合4参数logit曲线拟合,线性范围为1~170μg/L,灵敏度为0.52μg/L,检测限为1μg/L,饲料样、猪尿样的平均添加回收率分别为91.1%和89.2%,平均批内和批间变异系数〈15%,与多巴酚丁胺的交叉反应率为22.3%,与其他化合物无交叉反应,RAC-Kit在4℃可保存180d。Mixed anhydridewas used to conjugate ractopamine （RAC） to bovine serum albumin （BSA） and obtained artificial immunogen BSA-RAC identified byultraviolet and SDS-PAGE. Balb/C mice were immunized with BSA-RAC and hybridoma lines that secrete monoclonal antibody against RAC （RAC mAb） were generated with cell fusion. A blocking ELISA kit for detection RAC（RAC-Kit）was developed with RAC mAb and its traits were tested. The results showed that BSA-RAC was synthesized successfully and its conjugation ratio of RAC to BSA was about 24.5 ： 1. Three hybridoma lines were screened and the best one was 4D8, its affinity constant Ka was 1.65 × 10^10 L/mol. The calibration curve of RAC-Kit was typical sigmoid curve fitted to the 4-parameter logistic equation with the linear detection of 1 - 170 μg/L, the sensitivity of 0.52μg/L and the detection limit of 1μg/L. The recoveries of RAC spiked in feed were 91.1% and in swine urine were 89.2% . The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. RAC-Kit had 22.3% cross-reactivity to dobutamine and cross-reactivity to other compounds. The validity of RAC-Kit in 4℃ was above 180d.

关 键 词：莱克多巴胺 人工免疫原 单克隆抗体 阻断ELISA 快速检测试剂盒 

分 类 号：S859.84[农业科学—临床兽医学]