ATP柠檬酸裂解酶鼠源单克隆抗体的制备和鉴定

目的：制备ATP柠檬酸裂解酶（ACLY）鼠源单克隆抗体（monoclonal antibody，McAb），并进行相关的特异性鉴定。方法：该研究采用原核表达的重组蛋白ACLY为抗原免疫6~8周龄Balb/c雌性小鼠，利用杂交瘤融合细胞技术构建稳定分泌ACLY McAb的杂交瘤细胞，用酶联免疫吸附测定（ELISA）、免疫荧光（IF）、Western-blot、免疫组织化学（IHC）、流式细胞术（FCM）等鉴定其生物活性，检测所得抗体的特异性。结果：复筛后获得了一株能稳定分泌ACLY McAb的杂交瘤细胞（5F8D11），Ig亚类为IgG1，轻链为κ链；Western-blot、IHC、IF分析和ELISA检测证实该株ACLY McAb与ACLY具有较高的特异性。结论：该株抗ACLY特异性的McAb的成功制备，为检测ACLY蛋白表达水平及研究ACLY在肿瘤及心血管疾病中的致病机制提供有力的工具，将有助于对肿瘤及心血管疾病患者制定多样合理的治疗方案。

Preparation and identification of mouse-derived monoclonal antibodies against ATP citrate lyase

OBJECTIVE To prepare the mouse monoclonal antibody (McAb) against ATP Citrate Lyase (ACLY) and identify its specificity.METHODS The recombinant protein ACLY expressed in prokaryote was used as the antigen, and 6~8 week old Balb/c female mice were immunized with this protein ACLY.The hybridoma cells which stably secreting anti-ACLY McAb was constructed by using hybridoma fusion cell technology.The bioactivity of monoclonal antibody against ACLY was identified by enzyme linked immunosorbent assay (ELISA)、immunofluorescence (IF)、Western-blot、immunohistochemistry (IHC)、flow cytometry (FCM), et al.RESULTS A hybridoma cell (5F8D11) which can stably secrete anti-ACLY McAb was obtained, and the Ig subclass is IgG1, light chain κ chain.Western-blot, IHC, IFand ELISA test confirmed that this strain ACLY McAb had high specificity with ACLY.CONCLUSION A McAb specific against ACLY was successfully prepared, which provides a powerful detection tool for ACLY protein expression level and the detecting pathogenic mechanism of ACLY in tumor and cardiovascular disease, and will contribute to the development of diverse and rational treatment options for patients with tumor and cardiovascular disease.