论文部分内容阅读
目的:制备抗人内皮抑素的单克隆抗体(mAb),为深入研究内皮抑素的作用机制提供一种有用的试剂。方法:以毕赤酵母表达的内皮抑索为免疫原免疫BALB/c小鼠,采用B细胞杂交瘤技术,以间接ELISA法筛选分泌抗人内皮抑素的特异性mAb的杂交瘤细胞株,并诱生腹水。腹水中的mAb采用 Protein A Sepharose CL-4B进行纯化。以标准的抗Ig血清做ELISA,鉴定mAb的Ig类、亚类及型;用免疫印迹法鉴定mAb的特异性。结果:获得1株可分泌抗人内皮抑素mAb的杂交瘤细胞株4E7,该株杂交细胞分泌的mAb属于IgGl(λ型)。腹水mAb的效价为1×10-4-1×10-6,纯化后大于1×10-10。免疫印迹结果显示,mAb 4E7可特异性地与酵母及大肠杆菌表达的内皮抑素起反应,而与bFGF等其他细胞因子不发生交叉反应。结论:分泌抗人内皮抑素mAb杂交瘤细胞株的建立,为进一步研究奠定了基础。
Objective: To prepare anti-human endostatin monoclonal antibody (mAb), provide a useful reagent for further study of the mechanism of action of endostatin. Methods: BALB / c mice were immunized with endostatin of Pichia pastoris. The hybridoma cell lines secreting anti-human endostatin-specific mAb were screened by indirect ELISA with B cell hybridoma Ascites induced. MAbs in ascites were purified using Protein A Sepharose CL-4B. The standard anti-Ig serum ELISA, identification of mAb Ig class, subclass and type; using immunoblotting to identify the specificity of mAb. Results: One hybridoma cell line 4E7 secreting anti-human endostatin mAb was obtained. The mAb secreted by the hybridoma cells belonged to IgG1 (λ type). Ascites mAb titer of 1 × 10-4-1 × 10-6, greater than 1 × 10-10 after purification. Immunoblotting results showed that mAb 4E7 specifically reacted with endostatin expressed by yeast and E. coli and did not cross-react with other cytokines such as bFGF. Conclusion: The establishment of mAb secreting anti-human endostatin mAb hybridoma cell lines, laid the foundation for further study.