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目的:研制鼠抗人B7-H4分子的单克隆抗体(mAb)并鉴定其生物学特性。方法:以高表达人B7-H4分子的转基因细胞L929/B7-H4为免疫原,常规免疫BALB/c小鼠,采用B淋巴瘤杂交技术,经免疫荧光标记和流式细胞术分析、反复筛选、多次克隆化培养,获得两株可特异分泌鼠抗人B7-H4分子mAb的杂交瘤细胞株。采用快速定性试纸分析法鉴定mAb所属的Ig亚类。用Dot-blot、Western blot鉴定mAb的特异性,竞争结合抑制实验鉴定mAb所识别的抗原结合位点,T细胞增殖抑制阻断实验对mAb的生物学功能进行初步的鉴定。结果:获得两株持续、稳定地分泌抗B7-H4 mAb的杂交瘤细胞,分别命名为1F10和2B2。Dot-blot的结果显示,两株mAb均能特异性识别B7-H4分子。Westernblot检测显示,mAb 2B2可以特异性识别B7-H4分子,而mAb 1F10则不能识别。位点竞争实验表明,两株mAb之间,mAb 1F10与商品化的mAb H74之间识别位点不同,mAb 2B2与商品化mAb识别位点相近。T细胞增殖抑制阻断试验显示,这两株mAb均能一定程度地阻断B7-H4对T细胞的抑制作用。结论:成功地获得两株抗人B7-H4分子的mAb,为进一步研究B7-H4分子的生物学功能提供了有效的工具。
Objective: To develop a monoclonal antibody (mAb) against mouse anti-human B7-H4 and identify its biological characteristics. Methods: BALB / c mice were immunized with L929 / B7-H4, a transgenic B7-H4 cell line, which was overexpressed by human B7-H4. BALB / c mice were immunized with B lymphoma by immunofluorescence staining and flow cytometry. , Cloned and cultured for several times to obtain two hybridoma cell lines that can specifically secrete mouse anti-human B7-H4 molecule mAb. Identification of the Ig subclasses to which mAbs belong using rapid qualitative test strip analysis. The specificity of mAb was identified by Dot-blot and Western blot. Antibodies binding to mAb were identified by competitive inhibition assay. The biological function of mAb was preliminarily identified by T cell proliferation inhibition test. Results: Two hybridoma cells that consistently and stably secreted anti-B7-H4 mAb were obtained and named as 1F10 and 2B2, respectively. Dot-blot results showed that both mAbs specifically recognize B7-H4. Western blot analysis showed that mAb 2B2 specifically recognized the B7-H4 molecule, whereas mAb 1F10 did not recognize it. Site-directed competition experiments showed that there was a difference in recognition site between the two mAbs, mAb 1F10 and the commercial mAb H74, and mAb 2B2 was similar to the commercial mAb recognition site. T cell proliferation inhibition blocking test showed that both mAb can be blocked to a certain extent B7-H4 on T cell inhibition. Conclusion: The successful obtaining of two mAbs against human B7-H4 molecule provides an effective tool for further study on the biological function of B7-H4.