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目的:以本科室发现肿瘤细胞上表达的CD4078AA突变为基础,研制识别肿瘤细胞上CD40突变体分子的单克隆抗体(mAb),并对其生物学特性作初步分析。方法:以转人CD40突变体转基因细胞L929-CD40mu为免疫原,免疫6～8周龄的雌性BALB/c小鼠;采用B淋巴细胞融合技术,将免疫小鼠脾脏细胞与Sp2/0融合,以L929-CD40mu转基因细胞为抗体筛选阳性细胞,免疫荧光标记法对杂交瘤进行反复筛选和多次的克隆化培养;采用快速定性试纸法及竞争抑制结合试验分析该mAb的亚类及抗原识别位点;免疫印迹法对该mAb进行鉴定;采用MTT法分析mAb在体外对肿瘤细胞的抑制增殖效应以及PI-annexinⅤ方法进行细胞凋亡测定。结果:获得1株稳定分泌鼠抗人CD40mumAb的杂交瘤细胞株(命名为10C5),该mAb能特异性地识别人肿瘤细胞株HO8910表达的CD40突变体分子,而不识别正常扁桃体B淋巴细胞及血管内皮细胞表达的CD40分子,并且能够在体外促进肿瘤细胞凋亡。结论:成功地研制出1株特异性识别肿瘤细胞上CD40突变体分子的mAb,该mAb具有体外抑制肿瘤细胞生长并促进其凋亡的作用。
OBJECTIVE: To develop a monoclonal antibody (mAb) recognizing the CD40 mutant on tumor cells based on the discovery of the CD4078AA mutation in tumor cells in our hospital and to analyze its biological characteristics. Methods: Female BALB / c mice of 6-8 weeks old were immunized with L929-CD40mu transgenic mice transformed with CD40 mutant. The spleen cells of immunized mice were fused with Sp2 / 0 by B lymphocyte fusion technique, The positive cells were screened by L929-CD40mu transgenic cells and the hybridomas were screened repeatedly and cloned by immunofluorescence. The sub-class and antigenic recognition sites of the mAbs were analyzed by rapid qualitative test strip method and competitive inhibition binding assay The immunoblotting method was used to identify the mAb. MTT assay was used to analyze the inhibitory effect of mAb on tumor cells in vitro and PI-annexinⅤ method. Results: One hybridoma cell line stably secreting mouse anti-human CD40mumAb (named as 10C5) was obtained. The mAb specifically recognizes the CD40 mutant molecule expressed by human tumor cell line HO8910 without recognizing normal tonsil B lymphocytes and Vascular endothelial cells express CD40 molecules and are capable of promoting tumor cell apoptosis in vitro. CONCLUSION: A mAb that specifically recognizes CD40 mutant molecules on tumor cells has been successfully developed. The mAb has been shown to inhibit tumor cell growth and promote apoptosis in vitro.