文档介绍：鼠抗人TCR/CD3单克隆抗体的制备及其初步应用
(作者:___________单位: ___________邮编: ___________)
作者:陈宏远,芮雯,花文峰,杜军,蔡绍晖
【摘要】目的应用B淋巴细胞杂交瘤技术,制备鼠抗人CD3 单克隆抗体,并用于T淋巴细胞亚类检测。方法以本室纯化的CD3蛋白免疫Balb/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,成功地获得1株抗人CD3单克隆抗体。经间接ELISA测定,杂交瘤细胞培养腹水的抗体效价。结合SDSPAGE实验考察单克隆抗体对CD3促T淋巴细胞增殖活性的作用。结果杂交瘤细胞培养腹水的抗体含量为20 mg/mL,效价为10-7,并对T淋巴细胞有促增殖活性,检测正常人外周血阳性T细胞百分率为(73±7)%。结论制备的抗人CD3单克隆抗体具备较高的纯度和活性,可用于T细胞亚类的检测。
【关键词】 CD3;单克隆抗体;杂交瘤
Abstract: Objective To detect the CD3 positive T lymphocytes subgroup with the monoclonal antibody of mouse antihuman CD3 antigen of T lymphocyte which produced by B lymphocyte hybridoma technique. Methods Balb/c mice were immunized with CD3 protein produced in our laboratory, mixed SP2/0 myeloma with spleen cells of the mice, then obtained
an antihuman CD3 antigen of hybridoma cell line. The ascitic culture monoclonal antibody titer was determined by indirectELISA, the quantity and molecular weight were determined by SDSPAGE. MTT method was contributed to explore its activity of promoting T lymphocyte proliferation. Results The monoclonal antibody quantity of ascitic hybridoma cell was 20 mg/mL and the titer was 10-7. It had an activity of promoting T lymphocyte proliferation, percentage of T lymphocyte in healthy adult peripheral blood samples was (73±7)%.Conclusion This results suggest that the antiCD3 monoclonal antibody had high purity and activity that might be used in the detection of CD3 positive T lymphocytes subgroup, and could be applied in T lymphocyte detection.
Key words: CD3;monoclonal antibody; hybridomas
CD3是T淋巴细胞的一个分化抗原,表达于所有成熟T细胞表面,它是由5条肽链非共价结合组成的复合分子,分别称为γ、δ、ε、ζ和η链。其与TCR分子以非共价结合形成一个TCR/CD3复合受体分子,其中TCR是识别异种抗原和自身MHC分子多态性决定族的受体,而CD3分子并不参与抗原识别,它具有稳定TCR结构和传递活化信号的作用。TCR/CD3复合体是T细胞功能和特异性的主要调节者,抗TCR/CD3 单克隆抗体已在基础研究和临床中都被广泛应用。如何制备质量好、产量高的单克隆抗体是当前各有关实验室研究的重要问题。目前用于生产单克隆抗体的方法有2种:利用纯系小鼠生产免疫腹水法和杂交瘤细胞常规体外培养法[1,2]。鉴于本次试验样品备用于后续的研发,以及腹水法易保持生物学活性、耗时短和易操作等优点,故应用B淋巴细胞杂交瘤技术联合腹水法,制备并筛选了1株抗人CD3 单克隆抗体,并对其特性进行了鉴定和检测,获得较为满意的结果,现报道如下。
1 材料与方法
材料
纯