抗人BPI_(23)单克隆抗体的制备和鉴定  

Preparation and identification of anti-human BPI_(23) monoclonal antibody

作　　者：胡智颖[1] 万云霞[2] 郭素娟[2] 李蕴[1] 何秀娟[1] 龙军[1] 安云庆[1] 

机构地区：[1]首都医科大学免疫学系,北京100069 [2]中国医学科学院肿瘤医院肿瘤研究所免疫室,北京100021

出　　处：《基础医学与临床》2009年第10期1097-1101,共5页Basic and Clinical Medicine

基　　金：北京市自然科学基金(7082016)

摘　　要：目的采用杂交瘤技术制备抗人BPI23单克隆抗体,并对其应用进行初步分析。方法免疫小鼠脾细胞与小鼠骨髓瘤细胞按常规方法融合;用间接ELISA法和Western-blot筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法亚克隆3次获得稳定分泌抗人BPI23单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注入小鼠腹腔后制备腹水;纯化腹水中的单抗并对抗体类型进行鉴定;用Western-blot分析抗体的特异性;用间接ELISA法测抗体效价;将分离纯化的正常人外周血中性粒细胞和单个核细胞制成涂片,用抗人BPI23单克隆抗体进行免疫染色。结果获得3个(1B4、9C12和2H11)稳定分泌抗人BPI23单克隆抗体的杂交瘤细胞株,所分泌的单抗类型分别为κ型IgM、κ型IgG1和κ型IgG1;抗体效价分别为1.28×105、1.28×105和4.1×106,纯化后抗体含量分别为0.208g/L、2.03g/L和3.88g/L;3种纯化抗体均能与本实验制备的人BPI23和市售人BPI55标准品特异性结合,而不能与小鼠BPI25和人LBP结合;在免疫组化实验中,1B4、9C12和2H11单抗均能与人中性粒细胞中的BPI特异性结合。结论成功制备了人BPI23特异性单克隆抗体,为BPI检测试剂盒的研制奠定了基础。Objective Preparation of anti-human BPI23 monoclonal antibody with hybridoma technique and preliminary analysis of the application. Methods Theimmunized mouse spleen cells and mouse myeloma cells were fused by routine method; Indirect ELISA and Western-blot were used to screen the hybridoma cells;the positive clones were subcloned acquire hybridoma cells secreting anti-human BPI23 monoclonal antibody;the amplified hybridoma cells were injected into abdominal cavity of mouse and the ascites was collected;the antibodies in ascites were purified and titrated; the specificity of the antibody was analyzed by Western-blot; the normal human neutrophil and mononuclear cells were separated and purified for smear. Results Three hybridoma cell lines （1B4, 9C12 and 2H11）secreting anti-human BPI23 monoclonal antibody were acquired. The anti-human BPI23 monoelonal antibodies secreted by hybrldoma cells was kappa opioid IgM class , kappa opioid IgG1 subclass and karma opioid IgG1 subclass respectively;the titer was 1.28×10^5、1.28×10^5 and 4.1×10^6 respectively. Western-blot showed that me monoclonal antibody from ascites of tumor-bearing mice can recognize human BPI23 protein prepared in our lab and commercially available human BPI55 standards saled in the market specifically as well, but can＇t bind mouse BPI25 and human LBP;in immunohistochemistry. The 1B4, 9C12 and 211. monoclonal antibody can recognize BPI of human neutrophil specifically. Conclusion The monoclonal antibody against human BPI23 was prepared successfully,which lay a foundation for later establishment of BPI detecting kit.

关 键 词：人BPI23 杂交瘤细胞 单克隆抗体 

分 类 号：R392[医药卫生—免疫学]