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抗人红细胞膜抗原非凝集型单克隆抗体的研制及特性鉴定

发布时间:2019-01-11 08:54  文章来源:笔耕文化传播
【摘要】:抗人红细胞膜抗原非凝集型单克隆抗体(mAb)是与自身全血凝集试验相关的各类双特异性抗体分子的重要组成部分。以非凝集型单抗为基础的双特异性抗体分子可快速、准确地检测人体血液中感染病原体的抗原或抗体成分。 本研究应用杂交瘤技术,将人O型红细胞膜免疫BALB/c小鼠,取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞融合,聚凝胺试管法筛选出识别红细胞表面共同抗原的抗体,玻片法剔除凝集型抗体(IgM、IgA),再将分泌非凝集型抗体(IgG)的杂交瘤细胞株有限稀释法克隆3次,对杂交瘤细胞的稳定性和mAb的特性进行鉴定。 本研究获得1株可稳定分泌IgG1类mAb的杂交瘤细胞,命名为2E8。mAb2E8针对红细胞膜H抗原,没有血型交叉反应和种属交叉反应,细胞培养上清与人的A、B、AB和O型红细胞均能形成很强的凝集,凝集效价为1:1024,腹水凝集效价达到1:64×10~6。mAb2E8与红细胞结合后能诱导白细胞或激活补体,有效地破坏、溶解红细胞。此外,本研究建立了筛选针对红细胞表面抗原mAb的新方法—聚凝胺试验,为制备与红细胞相关的各种mAb的筛选提供一个简单快捷的途径。 本研究通过杂交瘤技术,成功地制备了针对红细胞膜H抗原的非凝集性mAb,并对抗体的各项指标进行了较全面的鉴定。该mAb的凝集效价、相对亲和力及特异性均良好,且具有较强的体外生物学活性,为构建与自身全血凝集试验相关的双特异性抗体分子和单抗异聚体(HP)奠定了坚实的基础。
[Abstract]:Anti-human erythrocyte membrane antigen non-agglutination monoclonal antibody (mAb) is an important component of all kinds of bispecific antibody molecules associated with autohemagglutination test. Bispecific antibody molecules based on non-agglutinating monoclonal antibodies can be used to detect the antigen or antibody components of infected pathogens in human blood quickly and accurately. In this study, BALB/c mice were immunized with human O type erythrocyte membrane by hybridoma technique. The spleen cells of immunized mice were fused with SP2/0 myeloma cells. The agglutination antibody (IgM,IgA) was eliminated by slide method. The hybridoma cell line which secreted non-agglutinative antibody (IgG) was cloned for 3 times by limited dilution method. The stability of the hybridoma cells and the characteristics of mAb were identified. In this study, we obtained a hybridoma cell line which can secrete IgG1 mAb stably, named 2E8.mAb2E8 for erythrocyte membrane H antigen, no blood group cross reaction and species cross reaction, cell culture supernatant and human Agna B, The agglutination titer of AB and O-type red blood cells was 1: 1024, and the agglutination titer of ascites reached 1:64 脳 10~6.mAb2E8, which could induce white blood cells or activate complement, effectively destroy and dissolve red blood cells. In addition, a new method for screening erythrocyte surface antigen (mAb) was established, which provides a simple and rapid way for the preparation of various mAb related to erythrocyte. In this study, nonagglutinating mAb, against erythrocyte membrane H antigen was successfully prepared by hybridoma technique, and various indexes of antibody were comprehensively identified. The agglutination titer, relative affinity and specificity of the mAb were good, and had strong biological activity in vitro, which laid a solid foundation for the construction of the bispecific antibody molecule and the monoantibody heteropolymer (HP) related to the whole blood agglutination test.
【学位授予单位】:中国人民解放军军事医学科学院
【学位级别】:硕士
【学位授予年份】:2005
【分类号】:R392

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