抗弓形虫主要表膜P30抗原单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against toxoplasma major surface antigen-P30

目的 制备抗弓形虫主要表膜P30抗原单克隆抗体 (McAb)并进行鉴定 ,为弓形虫病的诊断、抗原的提纯及亚单位疫苗的研制等提供可靠依据。方法 用RH株弓形虫速殖子膜抗原为免疫原免疫BALB/C小鼠 ,取其脾细胞与小鼠SP2 / 0骨髓瘤细胞融合 ,筛选出能够稳定分泌高滴度抗P30抗原McAb杂交瘤细胞株 ,并测定McAb免疫球蛋白亚类和单抗效价 ,用IFAT进行单抗识别的抗原定位 ,并通过SDS PAGE和Western blot分析鉴定。结果 本实验获得了 2株抗P30抗原的杂交瘤细胞株E3 和G2 ,分泌的抗体滴度分别为 1∶2 5 880和 1∶10 2 6 0 ,其分泌的抗体与肺孢子虫、隐孢子虫等抗原均不发生交叉反应 ,2株单抗均属IgG1亚类 ,且识别的抗原定位于速殖子表膜。

Objective: To develop and identify the monoclonal antibodies(McAb) against the major surface antigen P30 of Toxoplasma gondii, which may provide useful help for further study. Methods: BALB/c mice were immunized with the surface antigen of toxoplasma, and the spleen cells of immunized mice were fused with myeloma cell line SP2/0. After the cell fusion, positive hybridoma cell lines were screened by ELISA. Results: McAbs of E3 and G2 were identified to be against the major surface antigen P30 by SDS PAGE and Western Blot. These two clons of the McAbs were IgG1 subtypes by ELISA testing and the hybridoma cells could secrete higher titers of McAbs. The antigens recognized by McAbs by IFAT were located on the surface of tachyzoite. The McAbs had no cross reactions with antigens of Cryptosporidium、Pneumosystis caranii and Cysticercus cellucose. Conclusion: The hybridoma cell lines which can secrete high titers of anti P30 have been established.