抗人血红蛋白单克隆抗体的制备及鉴定

Preparation and identification of anti-hemoglobin monoclonal antibody

目的运用杂交瘤技术制备抗血红蛋白(hemoglobin,Hb)单克隆抗体,为进一步探讨Hb在帕金森病中的发病机制及早期诊断中的意义奠定基础。方法用重组人Hb作为抗原免疫Balb/c小鼠,并将其脾细胞与同系骨髓瘤细胞融合制备杂交瘤,经3次有限稀释法及酶联免疫吸附试验法筛选获得抗人Hb单克隆抗体杂交瘤株5B1。腹腔注射于免疫抑制Balb/c小鼠产生腹水,并利用Dot blotting、酶联免疫吸附实验,免疫共沉淀、Western blotting法及免疫组织化学染色法进一步测定抗体效价,获得一特异性抗Hb单克隆抗体。结果成功制备出1株高亲和力、特异性好的抗Hb单克隆抗体。结论制备的单克隆抗体对Hb具有特异性(包括人血、猴及小鼠),为进一步研究Hb在帕金森病(Parkinson's disease,PD)中的作用提供了有力的特异性工具。

Objective To prepare a specific antibody against human hemoglobin（ Hb） by hybridoma technique,which has great significance in further exploring the functions of Hb in early diagnosis and pathogenesis of Parkinson＇s disease. Methods The fusion cells were produced with spleen cells of two Balb / c mice immunized with recombinant human Hb to combine with homologous myeloma cells. A positive clone,5B1,was selected by screening the hybridoma cells with the enzyme-linked immunosorbent assay（ ELISA） after three times of limited dilution method. The antibody was acquired by intraperitoneal injection in immunosuppressed BALB / c mice. And Dot blotting,ELISA,Co-immunoprecipitation（ Co-IP）,Western blotting tests and immunohistochemical（ IHC） staining were used for further testing. Results One stable hybridoma cell line,named as 5B1,was found to secret anti-Hb with high titers and specificity. Conclusion The results demonstrated that the monoclonal antibody made in this study has specificity to Hb in human blood,monkey,rats and mouse,and offers a powerful tool for future studies on Hb in Parkinson＇s disease（ PD）.