金黄色葡萄球菌肠毒素Ⅰ单克隆抗体的制备和鉴定  

Preparation and identification of monoclonal antibodies to staphylococcal enterotoxin Ⅰ

作　　者：徐君英[1,2] 丁丁[1] 孙红颖[1] 陈枢青[1] 

机构地区：[1]浙江大学药学院药理毒理与生化药学研究所,浙江杭州310058 [2]北京中医药大学生物制药系,北京100102

出　　处：《浙江大学学报（医学版）》2009年第3期265-270,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省科技厅重大科技攻关项目(2004C13041)

摘　　要：目的:制备稳定分泌抗金黄色葡萄球菌肠毒素(SEI)单克隆抗体的杂交瘤细胞株,并对单克隆抗体的性质进行鉴定。方法:以SEI蛋白免疫Balb/c小鼠,利用细胞融合技术,经ELISA检测筛选及3次有限稀释,获得目的杂交瘤细胞株;采用杂交瘤细胞免疫小鼠,制备并纯化了单克隆抗体,并对所获得的单克隆抗体进行性质鉴定。结果:最终获得了两株稳定分泌抗体的杂交瘤细胞8F7与D8,两者分别为IgG2b型与IgG1型;亲和常数分别为9.215×108L.mol-1和1.968×1010L.mol-1;两者均有较强的特异性,与纯化的SEG、SEE、SEC及金黄色葡萄球菌上清液均未有交叉反应;相加实验表明两个单克隆抗体识别的为相同表位。结论:单克隆抗体的成功制备,为初步建立肠毒素SEI的双抗夹心测定方法奠定了基础。Objective： To prepare and identify monoclonal antibodies against staphylococcal enterotoxin Ⅰ （SEI）. Methods： Spleen cells obtained from mice immunized with the SEI protein were fused with the myeloma cells （SP2/0）. Hybridomas were screened by enzyme-linked immunosorbent assay （ELISA） and the stable monoclonal hybridomas were isolated by limiting dilution at least three times. The characters of purified monoclonal antibodies were identified by indirect ELISA and Western blotting. Results： The monoclonal antibodies secreted by two hybridomas 8F7 and D8 belonged to IgG2b and IgG1 subtypes. Both had high titer and specificity with no cross reaction to SEG,SEE and SEC. Conclusion： The monoelonal antibodies against SEIhas been successfully prepared and identified in this study.

关 键 词：肠毒素类/免疫学 葡萄球菌 金黄色/免疫学 抗体 单克隆 小鼠 近交BALBC 杂交瘤 酶联免疫吸附测定 免疫球蛋白G 

分 类 号：R392[医药卫生—免疫学]