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.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
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
关键词
重组可溶性组织因子
免疫
单克隆抗体
recombinant soluble tissue factor
immunity
monoclonal antibody
