关键词: 单克隆抗体, 制备技术, 半固体培养, 筛选鉴定策略, 恶性疟原虫, 富组氨酸蛋白

Abstract:

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(rePf-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 IgG 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.