β_2糖蛋白Ⅰ单克隆抗体的制备与鉴定  

Preparation and identification of the monoclonal antibodies against β_2-glycoprotein Ⅰ

作　　者：胥亚[1] 许国莹[2] 周红[1] 解鸿翔[1] 夏龙飞[1] 刘敬敬[1] 张晓蕾[1] 

机构地区：[1]江苏大学基础医学与医学技术学院,镇江212013 [2]淮阴卫生高等职业技术学校.淮安223300

出　　处：《中国免疫学杂志》2013年第6期649-652,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(No.30971301)资助

摘　　要：目的:制备β2糖蛋白Ⅰ(β2GPⅠ)单克隆抗体(MAbs),并鉴定抗体的特性。方法:以纯化的人血浆β2GPⅠ免疫BALB/c小鼠,采用PEG融合技术,间接ELISA法和有限稀释法,制备和筛选杂交瘤细胞。杂交瘤细胞继续扩大培养后注入BALB/c小鼠腹腔制备腹水,经硫酸铵沉淀及Protein G纯化MAbs。秋水仙素阻抑法鉴定杂交瘤细胞株染色体数目,ELISA测定滴度,Ig亚类试剂盒鉴定Ig亚类,Western blot鉴定特异性。结果:获得1株稳定分泌MAbs的杂交瘤细胞株β2,该杂交瘤细胞株的染色体数为99～108。进一步研究发现,其培养液和腹水滴度为1∶29和1∶215,其MAbs分类为IgG1/κ,Western blot显示纯化的MAbs及标准anti-β2GPⅠ与β2GPⅠ均有反应条带,具有较好的特异性。结论:成功获得并纯化β2GPⅠ单克隆抗体,为进一步研究β2GPⅠ/anti-β2GPⅠ复合物在抗磷脂综合征中的作用奠定了基础。Objective：To produce and identify the monoclonal antibodies （MAbs） against 132-glycoprotein I （132 GP I ）. Methods： The spleen cells from the BALB/c female mouse immunized with 132 GP I were fused with SP2/0 mouse myeloma cells in PEG to generate hybridoma cells, which were screened by limited dilution. Then the hybridoma cells were injected to BALB/c mice to make ascites, in which the antibodies were produced abundantly, followed by purified with ammonium sulfate and protein G. Colchi- cine was used to identify the number of chromosome of hybridoma cells. The properties of the MAbs against 132 GP I （ the titer of the MAbs in the cuhure medium and ascites, the isotypes of the MAbs and the specificity of the MAbs） were determined by indirect ELISA, MAbs isotyping kit and Western blot, respectively. Results： One hybridoma cell line （ 132 ）, secreting MAbs against 132GP I , had been established. The number of chromosome of the hybridoma ceils was between 99 and 108. Further studies found that the titer of the MAbs against 132GP I in the culture medium and ascites was 1：29 and 1：215 , respectively. The immunoglobulin of the MAbs was classified as IgG1/K, with satisfactory specificity. Conclusion： The MAbs against [32 GP I were prepared and identified, which pave the way for the research in the mechanism of antiphospholipid syndrome.

关 键 词：β2GPⅠ 杂交瘤细胞株 单克隆抗体 抗磷脂综合征 

分 类 号：R593.2[医药卫生—内科学]