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目的制备抗小鼠巨噬细胞移动抑制因子(mMIF)单克隆抗体(McAb)对其进行放射性碘标记,为进一步研究MIF在炎症、肿瘤核素显像中的意义奠定基础。方法异丙基硫代-β-D-半乳糖(IPTG)诱导pEQ30-mMIF/M15工程菌表达重组mMIF蛋白;采用杂交瘤技术制备单克隆抗体,蛋白质印迹技术(Western blotting)和酶联免疫吸附试验(ELISA)进行特异性鉴定,巨噬细胞移动抑制试验(MMI)鉴定生物学活性。采用二氯二苯基甘脲(Iodogen)法,用Na125I标记抗mMIF单克隆抗体。结果成功建立3株稳定高效价分泌抗mMIF单克隆抗体的杂交瘤细胞株,分别命名为5G2D7,5G2C7,5G2E3。经Western blotting、ELISA和MMI鉴定证明抗mMIF单克隆抗体具有良好的抗原特异性和生物学活性;125I-抗mMIF单克隆抗体的标记率为90.40%,放射化学纯度98.36%,放射性比活度为21.48MBq/μg。结论成功制备抗mMIF单克隆抗体和放射性碘标记抗mMIF单克隆抗体,为深入研究MIF相关性疾病和利用MIF进行临床肿瘤及炎症放射免疫显像提供了实验基础。
Objective To prepare a monoclonal antibody against murine macrophage migration inhibitory factor (mMIF) for radioactive iodine labeling, which will lay the foundation for the further study on the significance of MIF in imaging of inflammation and tumor. Methods Recombinant mMIF protein was induced by isopropyl thio-β-D-galactose (IPTG) -induced pEQ30-mMIF / M15 engineered bacteria. Monoclonal antibody, Western blotting and enzyme-linked immunosorbent assay Assay (ELISA) was performed for specificity, macrophage migration inhibition assay (MMI) for identification of biological activity. The anti-mMIF monoclonal antibody was labeled with Na125I using the Iodogen method. Results Three hybridoma cell lines secreting anti-mMIF monoclonal antibody were successfully established and named as 5G2D7, 5G2C7 and 5G2E3, respectively. The results of Western blotting, ELISA and MMI showed that anti-mMIF monoclonal antibody had good antigen specificity and biological activity. The labeling rate of 125I-anti-mMIF monoclonal antibody was 90.40% and the radiochemical purity was 98.36%. The radioactivity specific activity 21.48 MBq / μg. Conclusion The successful preparation of anti-mMIF monoclonal antibody and radioactive iodine labeled anti-mMIF monoclonal antibody provide experimental basis for further study of MIF-related diseases and clinical tumor and inflammatory radioimmunoimaging using MIF.