抗人类共激活蛋白单克隆抗体的制备  

Preparation of Monoclonal Antibody against Human Coactosin-like Protein

作　　者：蔡豪斌[1,2,3] 王丽兰[2] 何雨 詹庭芳 阳艳华 Eng-Shang Huang 

机构地区：[1]桂林英美特生物技术研究所,广西桂林541001 [2]桂林医学院临床检验系,广西桂林541004 [3]Linebege Cancer Center,University of North Carolina at Chapel Hill,usanc27599

出　　处：《中国生物制品学杂志》2009年第8期799-803,共5页Chinese Journal of Biologicals

摘　　要：目的制备抗人类共激活蛋白(CLP)单克隆抗体,并进行纯化。方法配制2×DMEM培养基,与等量的2.2%甲基纤维素溶液混合,加入HAT、胎牛血清等,配成半固体HAT选择性细胞培养基。用基因重组人CLP抗原免疫BALB/c小鼠,取免疫小鼠脾细胞,与小鼠骨髓瘤NS-2细胞融合后,直接在半固体培养基上克隆化培养,筛选可持续、稳定、高效分泌抗CLP单克隆抗体的杂交瘤细胞株。对分泌的单抗进行鉴定后,制备小鼠腹水,经DEAE离子交换柱纯化,半饱和硫酸铵沉淀,G25凝胶过滤除盐。结果共筛选出14株分泌抗CLP单抗的杂交瘤细胞,其培养上清可与相对分子质量约16000的CLP抗原结合,抗体类型为鼠IgGa1型。多数克隆分泌的抗体相对亲和力大于1∶20000,14株克隆分泌的单抗分别对应3个抗原结合位点。纯化后的单抗纯度接近95%。结论已成功制备并纯化了抗人CLP单克隆抗体,为建立CLP免疫学检测方法奠定了基础。Objective To prepare and purify the monoclonal antibody （McAb） against human coactosin-like protein （CLP）. Methods Mi× 2 DMEM with an equal volume of 2. 2% methylcellulose, then add HAT and fetal calf serum et al. to prepare semisolid HAT selective medium. The splenic cells of mice immunized with recombinant human CLP were fused with murine myeloma NS- 2 cells, then inoculated to the prepared medium for cloning culture. The hybridoma cell strains secreting McAb against CLP persistently, stably and effectively were screened, and the secreted McAbs were identified. BALB/c mice were injected i.p. with the screened hybridoma cells, and their aseites were purified by DEAE ion exchange chromatography and precipitated with 50% saturated ammonium sulfate, then filtrated with G25 gel for dechlorination. Results A total of 14 hybridoma cell strains secreting McAbs against CLP were screened, and their culture supernatants were bound to CLP antigen with a relative molecular mass of about 16 000. The secreted McAbs were murine IgGal. The relative affinities （RAs） of McAbs secreted by most of the cell strains were more than 1 ： 20 000. The McAbs secreted by the 14 strains were corresponding to 3 antigen binding sites, and reached purities of nearly 95% after purification. Conclusion The McAbs against CLP were successfully prepared and purified, which laid a foundation of developing a method for immunological detection of CLP.

关 键 词：类共激活蛋白 单克隆抗体 克隆化培养 

分 类 号：R392-33[医药卫生—免疫学] R730.21[医药卫生—基础医学]