鼠抗人内皮抑素单克隆抗体的制备与鉴定

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。

Preparation and identification of the monoclonal antibody against human endostatin

AIM: To prepare monoclonal antibody against human endostatin for the in depth study on the mechanism of anti-tumor effect of endostatin. METHODS: Monoclonal antibody specific for endostatin was prepared using hybridoma technique and screened with ELISA. Ascites fluids were produced in BALB/c mice following in sequentical intraperitoneal injection of pristine and hybridoma cells. The mAb was purified by affinify chromatography with protein A sepharose CL-4B. RESULTS: One hybridoma cell line 4E7 was established, which could produce mAb against human endostatin. The titers of mAb 4E7 in culture supernatant and ascites fluid were 1:128-1:516 and 1:10(-4)-1:10(-6), respectively. The mAb 4E7 belonged to IgG1 (lambda type). Western blot analysis showed that the mAb 4E7 could react to the human endostatin expressed by yeast and E.coli, but it had no cross reaction to other cytokines such as bFGF. CONCLUSION: The mAb 4E7 is able to react specifically with human endostatin and can be used for further investigation.