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目的:制备血管内皮生长因子(VEGF)单克隆抗体(mAb),并对其特性进行鉴定。方法:以重组His-Trx-VEGF1-165蛋白为抗原,通过免疫、融合、克隆和筛选等方法制备VEGF mAb,制备小鼠腹水并测定其效价、染色体数目及免疫球蛋白亚类,用Western blot方法分析其特异性。结果:成功筛选出能稳定分泌的VEGF mAb杂交瘤细胞株4E4-H5,制备腹水的ELISA效价为1∶106;染色体数目为105条;抗体分型为IgG1亚型,κ轻链;Western blot证实所获得的mAb可与VEGF全长蛋白发生特异性反应,但是与融合蛋白的标签蛋白His-Trx不发生反应。结论:制备的杂交瘤细胞株能稳定分泌高滴度和高特异性的VEGF mAb,为建立能够用于肝癌诊断的血清VEGF检测技术提供了参考。
Objective: To prepare the monoclonal antibody (mAb) of vascular endothelial growth factor (VEGF) and identify its characteristics. Methods: VEGF mAb was prepared by immunization, fusion, cloning and screening using recombinant His-Trx-VEGF1-165 as antigen. The mouse ascites was prepared and its titer, chromosome number and immunoglobulin subclass were determined. blot method to analyze its specificity. Results: The stable secreting VEGF mAb 4E4-H5 cell line was successfully screened. The titer of ascites was 1:106, the number of chromosomes was 105, and the antibody was classified as IgG1 subtype and κ light chain. Western blot The obtained mAb was confirmed to react specifically with the full-length VEGF protein but not with the His-Trx, a tagged protein of the fusion protein. CONCLUSION: The prepared hybridoma cell line can stably secrete high titer and high specificity of VEGF mAb, which provides a reference for establishing the serum VEGF detection technology which can be used for the diagnosis of liver cancer.