克百威单克隆抗体的制备及鉴定  

Preduction and Identification of Carbofuran Monaclonal Antibody

作　　者：朱江[1] 陆贻通[1] 

机构地区：[1]上海交通大学农业环境生态研究所,上海201101

出　　处：《科技通报》2005年第5期536-540,544,共6页Bulletin of Science and Technology

基　　金：国家自然科学基金资助项目(20337010);国家基础研究重大项目(2004CB418503);上海市基础研究重点项目(04JC14051)

摘　　要：本试验以人工合成的克百威免疫原免疫BALB/C F1小鼠,采用杂交瘤技术制备了5株能稳定分泌抗克百威单克隆抗体的杂交瘤细胞株。用培养扩增后的各建株细胞诱导小鼠生长腹水,腹水效价达10-6￣10-7。琼脂双扩试验表明各单抗蛋白均为IgG,抗体蛋白均为IgG2a亚类。用辛酸-硫酸铵沉淀法纯化单克隆抗体腹水,并测定抗体蛋白含量为3￣8 mg/mL;用SDS-PAGE进行纯度鉴定,电泳图谱均显示了IgG抗体及其重链和轻链条带,分离的抗体纯度在90%以上。对纯化的单抗5E2进一步做交叉反应试验,与其他氨基甲酸酯类杀虫剂残杀威、速灭威、灭多威、甲奈威、异丙威、仲丁威的交叉反应率分别为7.2%、4.5%、2.8%、7.9%、9.5%、8.9%,结果表明单抗5E2有较好的特异性。Five hybridoma cell lines secreting anti-BFNH McAbs were obtained by immunization of BALB/C F1 mice with the conjugates of Carbofuran immunogen, hybridoma technique and McAbs, and all categorized into IgG2a subtypes. These hybridomas were prepared ascites of the mice, and the titers of the McAbs in the ascites ranged from 10^-6 to 10^-7. McAbs in the ascites were purified by caprylic acid ammonium sulphate precipitation method. Reductive SDS-PAGE showed that each of purified McAbs displayed two bands, one is light chain of IgG molecule, and the other is heavy chain of IgG. No or few impurity protein bands. The analysis experiment of BFNH showed： Anti-BFNH McAb5E2 crossreacted with Propoxur, MTMC, Methomyl, Carbaryl, isopropenyl and BPMC, the cross reactivity were 7.2% ,4.5% ,2.8% , 7.9%,9.5% ,8.9% respectively, McAb5E2 was therefore considered interesting for developing a BFNH-specific ELISA.

关 键 词：克百威 单克隆抗体 杂交瘤技术 酶联免疫吸附测定法 

分 类 号：Q813.2[生物学—生物工程]