高亲和力莱克多巴胺单克隆抗体的研制及ciELISA检测方法的建立

  用混合酸酐法（MA）将莱克多巴胺（RAC）偶联于牛血清白蛋白（BSA）,合成人工抗原BSA-RAC,用UV和SDS-PAGE鉴定;用BSARAC免疫Balb/c小鼠,细胞融合技术建立高亲和力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ciELISA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24.5∶1;筛选出3株杂交瘤细胞,其中最好的4D8株亲和常数（Ka）为1.65×1010L/mol; RAC-Kit的检测限为0.5ng/ml,检测范围为0.5～151ng/ml,饲料样、猪尿样的平均添加回收率为87.2%,89.4%,平均批内和批间变异系数小于15%,与多巴酚丁胺的交叉反应率（CR%）为9.7%,与其它化合物无CR,RAC-Kit在4 ℃保存期为180d。

Generation of High Affinity Monoclonal Antibody and Development of ciELISA Kit for Rapid Detection of Ractopamine

Mixed anhydride(MA) was used to conjugate ractopamine (RAC) to bovine serum albumin(BSA) and obtained artificial antigen BSA-RAC identified by UV and SDS-PAGE. Balb/c mice were immunized with BSA-RAC and hybridoma lines that secrete RAC monoclonal antibody(mAb) were generated with cell fusion. A ciELISA kit for detection of RAC (RAC-Kit) was developed with RAC mAb and its traits were tested. The results indicated that BSA-RAC was synthesized successfully and its conjugation ratio of RAC to BSA was about 24.5∶1. Three hybridoma lines were filtered and the best one was 4D8, its affinity constant (Ka) was 1.65×1010L/mol. The detection limit of RAC-Kit was 0.5ng/ml and its detection range was 0.5 to 151ng/ml. The recoveries of RAC spiked in feed were 87.2% and in swine urine were 89.4%. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. It had 22.3% cross-reactivity (CR%) to dobutamine and little or no CR to other compounds. The validity of RAC-Kit in 4℃ was above 180d.