半固体培养及快速筛选策略在恶性疟原虫相关单克隆抗体制备中的应用

Application of Semi-solid Culture and Rapid Screening Strategy in the Preparation of Monoclonal Antibody against Plasmodium falciparum Histidine-rich Protein

目的以恶性疟原虫富组氨酸蛋白单克隆抗体的制备为例,分析半固体培养技术和筛选鉴定策略在单克隆抗体制备中的关键作用。方法用恶性疟原虫富组氨酸蛋白重组抗原(re Pf-HRP)免疫BALB/c小鼠,将免疫小鼠的脾细胞与小鼠骨髓瘤SP20细胞融合,于含2%甲基纤维素的半固体培养基中培养,使融合细胞呈集落化生长;将单细胞集落转移至液体培养基中培养;建立不同的抗原和ELISA检测方法对培养上清进行筛选;经粗筛和特异性筛选为阳性的细胞株置液氮保存。冻存复苏后,对稳定分泌特异性抗体的单抗细胞株进一步分析腹水产量、抗体亚类、抗体识别位点、抗体的亲和常数及配对检测抗原的灵敏度。结果细胞融合后经半固体培养获得的单克隆细胞株为915个,粗筛的克隆阳性率为37.8%(346/915);特异性筛选的克隆阳性率仅占粗筛检测阳性的2.6%(9/346),占总克隆数的0.98%(9/915),经液氮冻存复苏鉴定,获得了8株稳定分泌特异性抗体的单抗细胞株;经腹水产量、抗体亚类、抗体识别位点、抗体的亲和常数及配对检测抗原的灵敏度等检测,最终获得了2株可用于快速检测恶性疟患者血液样本中特异性循环抗原的配对抗体。结论半固体培养保证了可供筛选的融合细胞的数量,粗筛和特异性筛选结合的策略保证了快速筛选出特异性克隆。

Objective To prepare the monoclonal antibody of Plasmodium falciparum histidine-rich protein, and analyze the roles of semi-solid culture technique and screening strategies in the preparation of monoclonal antibodies.Methods BALB/c mice was immunized with the recombinant antigen of Plasmodium falciparum histidine-rich protein（re Pf-HRP）. The spleen cells of immunized mice were fused with SP20 cells. The fused cells were cultured in semi-solid medium containing 2% methylcellulose to promote colony growth. The single cell clone was transferred to a liquid medium. Testing methods for screening culture supernatant was established based on the immune antigen and other related antigens. The positive cell clones by coarse screening and specific screening were preserved in liquid nitrogen.The positive cell lines were used for ascite antibody preparation, identification of Ig G subclass, recognition sites,antibody affinity and application analysis on sensitivity of detecting antigen. Results A total of 915 cell clones were obtained in semi-solid culture after cell fusion. The positive rate by coarse screening was 37.8%（346/915）. The positive rate of specific screening accounted for only 2.6%（9/346） of the coarse screening-positive clones, 0.98%（9/915） of the total number of clones. Eight specific antibody-secreting cell clones were obtained after liquid nitrogen frozen recovery tests. After further detection, 2 specific cell clones could be used as a pair of antibody for rapid detecting circulating antigen in the blood of patients with falciparum malaria. Conclusion Semi-solid culture method can provide enough fused cells for screening. Combined strategy of coarse and specific screening ensures the rapid selection of specific clones.