论文部分内容阅读
目的:制备有生物学活性的抗人细胞间黏附分子-1(ICAM-1)单克隆抗体(mAb)并进行特性鉴定。方法:以纯品ICAM-1为抗原免疫BALB/c小鼠4次。采用杂交瘤技术,经3次亚克隆筛选稳定分泌抗人ICAM-1 mAb的杂交瘤细胞株。采用动物体内诱生的方法大量制备mAb。以protein G对其进行纯化后,用间接ELISA法测定mAb的效价并鉴定其Ig亚类。用Western blot鉴定mAb的特异性。结果:筛选出4株可稳定分泌抗人ICAM-1 mAb的杂交瘤细胞株,分别命名为B9株、F1株、C11株和D6株。4株mAb的Ig亚类均为IgG1。4株mAb培养上清的效价均为1∶1 000;腹水的效价B9株与D6株为1∶2×105,F1株与C11株为1∶4×105。纯化后mAb的蛋白浓度为1.2 g/L,均可与ICAM-1特异性结合。结论:成功制备出效价高、特异性良好的4株抗ICAM-1 mAb,为进一步研究ICAM-1的生物学功能和临床应用奠定了基础。
OBJECTIVE: To prepare and characterize a monoclonal antibody against human intercellular adhesion molecule-1 (ICAM-1) with biological activity. Methods: BALB / c mice were immunized 4 times with pure ICAM-1 as antigen. Hybridoma technology was used to screen the hybridoma cell lines stably secreting anti-human ICAM-1 mAb after 3 subclones. MAb was prepared in large quantities using animal in vivo methods. After purification with protein G, the mAb titers were determined by indirect ELISA and their Ig subclasses were identified. The specificity of mAb was identified by Western blot. Results: Four hybridoma cell lines secreting anti-human ICAM-1 mAb were selected and named as B9, F1, C11 and D6, respectively. 4 mAb Ig subclass are IgG1.4 mAb culture supernatant titers were 1: 1 000; ascites titer B9 and D6 1: 2 × 105, F1 and C11 strain was 1 : 4 × 105. After purification, the protein concentration of mAb was 1.2 g / L, which could specifically bind to ICAM-1. CONCLUSION: Four anti-ICAM-1 mAbs with high titer and good specificity were successfully prepared, which laid the foundation for further study on the biological function and clinical application of ICAM-1.