抗人肝素酶单克隆抗体的制备与鉴定

Preparation of the Monoclonal Antibody Against the Human Heparanase

目的:肝素酶在白细胞游走和恶性肿瘤转移的过程中发挥重要作用,肝素酶抗体的制备对于自身免疫病和肿瘤的良恶性鉴别诊断具有重要意义。制备抗人肝素酶单克隆抗体,用于肝素酶的研究及临床恶性肿瘤的鉴别诊断。方法:通过杂交瘤技术将分泌抗人肝素酶单抗的小鼠B细胞与小鼠骨髓瘤细胞Sp2/0融合,获得稳定分泌抗人肝素酶单抗的杂交瘤细胞;用有限稀释法获得单克隆,以重组人肝素酶及含肝素酶的血小板裂解液对抗体进行Western印迹检测。结果:Western印迹结果显示制备的单抗与人肝素酶具有特异性免疫识别特性。结论:获得了能够特异性免疫识别人肝素酶的分泌性抗人肝素酶单克隆抗体。

Objective： Mammalian endoglycosidase heparanase is primarily responsible for cleaving heparan sulfate proteoglycans present on the basement memberane of cells and extracellular matrix. It could be important in embryonic development and tumor metastasis. To preparate the monoclonal antibody against the human heparanase for further research of clinical diagnosis of the tumour. Methods： Hybridoma technique was used. The splenocytes of immunized mice were fused with myeloma cells Sp2/0 and selectively cultured by HAT medium. Hybridoma ceils were screened by indirect ELISA and limited dilution was used to clone. Results： One hybridoma cell line stably secreting a specific monoclonal antibody against heparanase was obtained and identified. Western blotting and ELISA showed that the monoclonal antibody had a specific affinity for the heparanase. Conclusion： The monoclonal antibody obtained revealed a potential perspective for tumor diagnostic.