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为了制备鼠抗人TLT-2单克隆抗体(mAb)并对其生物学特性进行初步的研究。以TLT-2基因转染细胞株免疫BALB/c小鼠,采用杂交瘤技术制备鼠抗人TLT-2 mAb,收集腹水,并用ProteinG亲和层析法纯化。以流式细胞术、IP和Western blot等方法鉴定该单抗的特异性。并通过流式细胞术、CCK8和ELISA等方法对单抗的生物学特性进行了初步分析。结果:获得一株持续稳定分泌鼠抗人TLT-2 mAb的杂交瘤细胞株,命名为8C10。杂交瘤细胞分泌的mAb的Ig亚类(型)为IgG1(κ)。IP和Western blot分析证明,8C10能特异性识别人TLT-2分子。经8C10荧光染色后流式细胞术的结果表明:TLT-2在T细胞表面呈弱阳性表达,在单核细胞和B细胞表面呈强阳性表达。细胞增殖实验结果显示,该mAb对T细胞的增殖有抑制作用。提示,成功获得了一株特异性鼠抗人TLT-2 mAb,该mAb对T细胞体外增殖及其细胞因子IFN-γ、IL-2、和IL-10的分泌均有明显的抑制作用。
In order to prepare mouse anti-human TLT-2 monoclonal antibody (mAb) and preliminary study of its biological characteristics. BALB / c mice were immunized with the TLT-2 gene-transfected cell line and murine anti-human TLT-2 mAb was prepared by hybridoma technique. The ascites was collected and purified by ProteinG affinity chromatography. The specificity of the McAb was identified by flow cytometry, IP and Western blot. The biological characteristics of mAb were analyzed by flow cytometry, CCK8 and ELISA. Results: Obtain a stable and stable secretion of mouse anti-human TLT-2 mAb hybridoma cell line, named 8C10. The Ig subclass (type) of the mAbs secreted by the hybridoma cells is IgG1 (κ). IP and Western blot analysis proved that 8C10 can specifically recognize human TLT-2 molecule. The results of flow cytometry after 8C10 fluorescent staining showed that TLT-2 was weakly positive on T cell surface and strongly positive on monocytes and B cell surface. The results of cell proliferation experiments show that the mAb can inhibit the proliferation of T cells. The results showed that a specific murine anti-human TLT-2 mAb was successfully obtained. The mAb could significantly inhibit the proliferation of T cells and the secretion of IFN-γ, IL-2 and IL-10.