用多糖-破伤风类毒素结合苗制备抗流行性脑膜炎球菌多糖单克隆抗体  

Preparation of Monoclonal Antibody Against Neisseria Meningococcal Serogroup A Polysaccharide(GAMP) by Using the Conjugate Vaccine of both GAMP and Tetanus Toxoid

作　　者：李佩珊[1] 张春燕[2] 李时君[1] 陈妍[2] 项美娟[1] 李方和[2] 张洪[1] 

机构地区：[1]武汉大学第一临床学院药剂科,430060 [2]华中科技大学同济医学院附属同济医院实验医学研究中心,武汉430030

出　　处：《医药导报》2011年第6期709-712,共4页Herald of Medicine

摘　　要：目的 研制抗A群奈瑟脑膜炎球菌荚膜多糖单克隆抗体（GAMP mAb）.方法 以A群奈瑟脑膜炎球菌荚膜多糖-破伤风类毒素（GAMP-TT）耦联物免疫BALB/c小鼠,用常规细胞融合与克隆化技术获得一株能稳定分泌抗GAMP mAb的杂交瘤细胞株（2E7）.采用秋水仙素阻断法测定其染色体数目,降植烷诱导法制备含该抗体的小鼠腹腔积液,辛酸-硫酸铵沉淀法纯化单克隆抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）灰度扫描测定提取物纯度,改良过碘酸盐氧化法制备辣根过氧化物酶（HRP）标记抗GAMP mAb,并采用中和法及其抗原替代法对抗GAMP mAb的特异性进行初步的鉴定.结果 所获得的杂交瘤细胞（2E7）具有很好的生长特性,染色体数目约为139.73条;抗体分泌量适中,Ig亚类为IgG1,提取物浓度1.76～2.32 mg·mL-1,纯度97.2%,标记抗GAMP mAb的效价为1：25 600;抗原替代实验与中和实验对其特异性考核,初步结果证实2E7 mAb具有很好的抗GAMP特异性.结论 成功制备抗GAMP mAb,为GAMP-TT疫苗的生产监测及产品质量控制提供了必要的物质基础.Objective To prepare monoclonal antibodies against Neisseria meningococcal serogroup A polysaccharide （GAMP）. Methods BALB/c mouse was imnmnized by GAMP-Tetanus Toxoid （TT）. Hybridoma cell lines secreting monoclona/antibodies against GAMP were screened after the fusion of mouse splenic ceils with SP2/0 cells. The number of chromosome in hybridoma cell was tested by colchicines-intereeption, and the ascite of mice was prepared by pristane-inducing method. The mAb was purified by octansic acid-ammonium sulfate precipitation,and purity of the extracted protein was measured by gray scale-scanning assay of SDS-PAGE. The specificity of anti-GAMP mAb was identified by neutration of antigen and antigen-substitute method. Results The hybridoma （2E7） had a healthy growth state, and the chromosome of which was 139.73. It produced proper amount of mAb, which was identified to be IgG1 The concentration of extracts was 1. 76- 2.32 mg· mL-1 ,and the purity was 97.2%. The potency of HRP-marked anti-GAMP mAb was 1 ： 25 600, and the anti-GAMP E7 mAb showed a specificity for GAMP by antigen substitution and neutralization assay. Conclusion The anti-GAMP E7 mAb has been successfully obtained, and it makes a necessary foundation for the quality control and manufacture monitoring during preparation of the GAMP-TT vaccine.

关 键 词：菌苗 多糖抗体 A群奈瑟脑膜炎球菌荚膜多糖 结合苗 单克隆抗体 

分 类 号：R979.5[医药卫生—药品] R943[医药卫生—药学]