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

Preparation and characterization of non-agglutinating mAbs against membrane antigen on human erythrocytes

目的:制备抗人红细胞膜抗原的非凝集型单克隆抗体(mAb)并鉴定其特性。方法:应用杂交瘤技术,以人O型红细胞膜抗原免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,用聚凝胺试管法筛选识别红细胞表面共同抗原的抗体,玻片凝集实验剔除凝集型抗体(完全抗体),再将分泌非凝集型mAb(不完全抗体)的杂交瘤细胞株用有限稀释法克隆化3次。对杂交瘤细胞的稳定性和mAb的特性进行鉴定。结果:获得1株可稳定分泌mAb的杂交瘤细胞2E8。mAb2E8为IgG1类,可特异性地识别红细胞膜上的H抗原,没有种属交叉血凝反应。杂交瘤细胞的培养上清与人的A、B、AB和O型红细胞均能产生强凝集,凝集效价为1∶1024,腹水mAb的凝集效价达到1∶64×106。mAb的亲和力用凝集试验检测,出现血凝的时间为7s,3min以内凝块>1mm。结论:成功地制备了针对红细胞膜H抗原的非凝集性mAb,此mAb的凝集效价、相对亲和力及特异性均较良好,为构建双特异性抗体奠定了基础。

AIM: To prepare monoclonal antibodies (mAbs) against membrane antigens on human erythrocytes and characterize their properties. METHODS: BALB/c mice were immunized with the membrane antigens of human type O erythrocytes. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. The antibodies to common antigen on human erythrocytes were screened by hexadimethrine bromide (polybrene) test tube method and then the agglutinating antibodies (complete antibodies) were weeded out by slide hemagglutination assay. The hybridoma cells secreting non-agglutinating antibodies (incomplete antibodies) were cloned by limiting dilution method. The stability of the obtained hybridoma cells and the properties of the mAbs were identified. RESULTS: One hybridoma cell 2E8 was obtained, which secreted non-agglutinating antibody. The mAb 2E8 belonged to IgG1, could agglutinate H antigen, and had no species cross-agglutination reaction. The titers of culture supernatant and ascites of 2E8 were 1∶1 024 and 1∶64×10 6 respectively. When the affinity of mAb 2E8 was evaluated by agglutination reaction, erythrocytes began to agglutinate after 7 seconds and the clots exceed 1 mm 2 in 3 minutes. CONCLUSION: The non-agglutinating mAb against H antigen was prepared successfully. The mAb 2E8 has good titer, affinity and specificity, which lays the foundation for preparation of bispecific antibodies (BsAb).