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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。
Objective: To prepare non-agglutinating monoclonal antibody (mAb) against human erythrocyte membrane antigen and characterize it. Methods: BALB / c mice were immunized with human O-type erythrocyte membrane antigen using hybridoma technique. The spleen cells of the immunized mice were fused with Sp2 / 0 myeloma cells, the antibody recognizing the common antigen on the surface of erythrocytes was screened by polybrene tube test, the agglutination antibody (complete antibody) was removed by slide agglutination test, and the non-agglutinated Hybridoma cell lines of mAbs (incomplete antibodies) were cloned 3 times by limiting dilution. Hybridoma cell stability and mAb properties were identified. Results: 1 hybridoma cell line 2E8 which can stably secrete mAb was obtained. mAb2E8 IgG1 class, can be specifically identified on the red blood cell membrane H antigen, there is no cross-species hemagglutination reaction. The hybridoma cell culture supernatant and human A, B, AB and O type red blood cells can produce strong agglutination, agglutination titer 1:1024, ascites mAb agglutination titer reached 1:64 × 106. The affinity of mAb was detected by agglutination test, the time of hemagglutination was 7s and the clot> 1mm within 3min. CONCLUSION: The non-agglutinating mAb against erythrocyte membrane H antigen was successfully prepared. The agglutination titer, relative affinity and specificity of this mAb were good, which laid the foundation for the construction of bispecific antibody.