抗D-二聚体单克隆抗体的制备和鉴定

发布时间：2018-04-11 10:21

本文选题：血栓性疾病 + D-二聚体　；参考：《湖北中医药大学》2011年硕士论文

【摘要】：研究目的：血栓性疾病已经成为临床上十分常见的疾病,严重威胁着人类的生命健康,近年来其发病率渐增且发病年龄呈年轻化之势,成为当代国内外医学界研究的热点和重点之一。制备出具有效价高、纯度高、特异性强、无交叉反应、重复性好以及可无限量生产等多项优点的抗D-二聚体单克隆抗体,可以使血栓性疾病的早期诊断、抗体导向血栓显像和抗体导向溶栓等更加快速、准确、简便。方法：本实验以人源性D-二聚体标准品作为抗原常规免疫BALB/c雌鼠,取免疫小鼠的脾细胞与同系骨髓瘤细胞SP2/0作电融合,HAT选择培养出杂交瘤细胞。以D-二聚体标准品为抗原,间接ELISA法筛选出分泌抗D-二聚体抗体的杂交瘤细胞,有限稀释克隆化培养获得稳定分泌抗D-二聚体单克隆抗体的阳性杂交瘤细胞株。利用ELISA、Western blotting等方法鉴定抗D-二聚体单克隆抗体的特性。结果：研究筛选出2株稳定分泌抗D-二聚体的单克隆抗体杂交瘤细胞株,分别命名为6A3和8A9。2株单克隆抗体的免疫球蛋白亚类均为IgG2a。通过ELISA、Western blotting等方法的鉴定,抗D-二聚体单克隆抗体能与D-二聚体发生特异性反应。结论：本实验成功制备出亲和力强、效价高,并且能特异性识别D二聚体的单克隆抗体,将为血栓性疾病的诊疗发挥重要作用。
[Abstract]:Objective: thrombotic disease has become a very common disease in clinic, which is a serious threat to human life and health. In recent years, the incidence of thrombotic diseases is increasing and the age of onset is becoming younger.It has become one of the hotspots and emphases of medical research at home and abroad.The preparation of anti-D-dimer monoclonal antibodies with the advantages of high effective value, high purity, strong specificity, no cross reaction, good repeatability and unlimited production can enable the early diagnosis of thrombotic diseases.Antibody-guided thrombolysis imaging and anti-thrombolytic imaging are more rapid, accurate and simple.Methods: in this experiment, BALB/c female mice were immunized with human D-dimer as antigen. The spleen cells of immunized mice and homologous myeloma cells SP2/0 were used as electrofusion fusion cells to culture hybridoma cells.The hybridoma cells secreting anti-Ddimer antibody were screened by indirect ELISA method using standard D- dimer as antigen. The hybridoma cell lines secreting monoclonal antibodies against D- dimer were obtained by limited dilution clone culture.The characteristics of anti-D-dimer monoclonal antibody were identified by Elisa-Western blotting.Results: two monoclonal antibody hybridoma cell lines which secreted anti-Ddimer stably were selected. The immunoglobulin subclasses of monoclonal antibodies named 6A3 and 8A9.2 were IgG2a respectively.The monoclonal antibody against D- dimer can react specifically with D- dimer by Elisa Western blotting.Conclusion: the monoclonal antibody with strong affinity, high titer and specific recognition of D dimer will play an important role in diagnosis and treatment of thrombotic diseases.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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