氨基脲单克隆抗体的制备及初步应用.pdf

中国动物检疫2009.9 氨基脲单克隆抗体的制备及初步应用 姚嘉赟 ，沈锦玉* ，郝贵杰，徐洋，潘晓艺,尹文林 （浙江省淡水水产研究所，浙江湖州 313001 ） 【摘要】 【目的】：制备抗氨基脲的单克隆抗体并建立ELISA检测方法。【方法】：以氨基脲 （SEM ）和对醛基苯 甲酸（CP ）为原料合成半抗原（CP-SEM ）,将半抗原和载体蛋白偶联后免疫Balb/C小鼠，应用杂交瘤技术建立稳定 分泌抗氨基脲的杂交瘤细胞株。常规制备腹水，用辛酸-硫酸铵法纯化，并对纯化的mAb进行特异性鉴定。通过对不 同抗体组合的分析和条件的优化，建立检测氨基脲的间接竞争ELISA方法。利用对醛基苯甲酸衍生草鱼肌肉提取的氨 基脲，用建立的ELISA方法进行检测，并计算回收率和最低检测限。【结果】：经细胞融合、筛选及克隆化，共获得7 株稳定分泌抗氨基脲的杂交瘤细胞株，其中4株亲和力较高。建立了间接竞争ELISA检测方法，该方法灵敏度达到 0.1ng/mL ，平均回收率为98.47％，在组织中的最低检测限为0.76 ng/g,回收率为70.55-100.56% ，可用于肌肉中氨基 脲残留的检测。【结论】：成功地制备了抗氨基脲的单克隆抗体，并建立了检测呋喃西林残留物氨基脲的间接竞争 ELISA法。 【关键词】：氨基脲 ；人工抗原 ；单克隆抗体；间接ELISA Preparation and primary application of monoclonal antibody against semicarbazide YAO Jia-yun,SHEN Jin-yu,HAO Gui-jie,XU Yang,PAN Xiao -yi,YIN Wen-lin Institute of Freshwater Fisheries of Zhejiang, Huzhou 313001, China [Abstract] Objective: To develop monoclonal antibody against semicarbazide and to establish a indirected ELISA method for quantitative analysis of semicarbazide .Methods: The hapten (CP-SEM) which was derivatived by combining 4-carboxybenzaldehyde(CP) with semicarbazide (SEM) was conjugated with carrierprotein to form complete antigen and then was used to immunize Balb/C mice ．Hybridoma cells secrecting mAb against semicarbazide were developed ．Ascites fr om the immunized mice were purified by caprylic acid-ammonium sulfate precipitation ．The specificity of the purified antibodies was detected and the suitable antibodies were used for establishing indirected ELISA. Results: Seven hybridoma cells stably secrecting mAb were prepared through cell fusion ，screening and cloning ， and four of these purified mAb had relative high aff inity ．The indirected ELISA for quantitative analysis of semicarbazi