脾内微量注射免疫法制备抗重组可溶性组织因子单克隆抗体  

Preparation of anti-recombinant soluble tissue factor monoclonal antibody by trace injecting antigen into spleen

作　　者：蔡旭[1] 彭卓醇[1] 孔德升[1] 郭泓珅[1] 白浩[1] 宋后燕[1] 马端[1] 

机构地区：[1]复旦大学上海医学院生物化学与分子生物学系-分子医学教育部重点实验室,上海200032

出　　处：《复旦学报（医学版）》2006年第1期92-96,共5页Fudan University Journal of Medical Sciences

基　　金：教育部科学技术重点项目(105064);上海市重大科技攻关项目(04DZ19104)资助

摘　　要：目的制备抗重组可溶性组织因子(r-sTF)单克隆抗体并建立检测组织因子(TF)含量的夹心ELISA法,以期为TF的深入研究及临床应用提供参考依据。方法取微量TF对Balb/c小鼠直接进行脾内注射免疫,分离小鼠脾细胞和鼠骨髓瘤细胞,融合后对杂交瘤细胞进行筛选克隆化培养;建立了3株稳定分泌TF单克隆抗体的杂交瘤细胞株,分别命名为1-C3、2-G9、3-H9;进而在小鼠体内诱生腹水,并用rprotein A亲合层析法纯化;经鉴定后用过碘酸钠法将辣根过氧化物酶标记于其中一株单抗上,建立夹心ELISA法测定正常人血浆中的TF含量。结果纯化的单抗用SDS-PAGE测定纯度,在Mr为150 000左右呈现一条带;Western blot测定显示1-C3、2-G9、3-H9分泌的单抗可特异地识别Mr为47 000的TF,经免疫球蛋白(Ig)类和亚类鉴定实验证实3株单抗均为IgG1;用夹心ELISA法检测正常人血浆中的TF含量为(130±80)pg/mL。结论用脾内微量注射法成功制备抗TF单克隆抗体及ELISA测定TF含量方法的建立,为TF的制备纯化和检测奠定了基础,也使TF的临床深入化研究成为可能。Purpose To prepare the anti-recombinant soluble tissue factor monoclonal antibody to develop an ELISA method which can be used to detect the tissue factor （TF） ; and to give a reference for going deep into the research and clinical application of TF. Methods We used TF to immunize Balb/ c mice by injecting TF into the spleen directly. Then the spleen cells were separated and fused with mouse myeloma cells（SP2/0） to obtain the hybridoma. The hybridoma was cloned and got 3 clones named as 1-C3, 2-G9, and 3-H9 which could stably secrete TF, and then they were embedded in mice bodies to induce ascites. TF was purified by protein A affinity chromatograph column and labeled with HRP. At last, normal humans＇ plasma TF levels were determined by ELISA developed from the antibody. Results The purified TF identified as IgG1 was a single band about Mr150 000 by SDS-PAGE analysis and Western blot showed that it could specifically recognize TF. Normal humans＇ plasma TF was about （130 ± 80）pg/mL as measured by ELISA. Conclusions We have prepared monoclonal antibody against TF and developed ELISA assays for TF successfully. The research will be useful for TF purification, measurements and clinical study.

关 键 词：重组可溶性组织因子 免疫 单克隆抗体 

分 类 号：Q813.1[生物学—生物工程]