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目的制备抗恶性疟原虫EBA-175的单克隆抗体(McAb),为恶性疟原虫的诊断及疫苗研究奠定基础。方法以恶性疟原虫EBA-175(II区F2段)重组抗原免疫BALB/c小鼠,采用杂交瘤技术制备McAb,间接ELISA筛选分泌高滴度McAb的杂交瘤细胞株,测定其免疫球蛋白亚类及其效价,ELISA、Westernblot试验分析其特异性。结果筛选获得6株稳定分泌抗重组EBA-175McAb的杂交瘤细胞株1F3、2E8、2H5、4A1、4C3、4H9,6株McAb经鉴定其亚类5株为IgG1,1株为IgG2a,6株McAb的培养上清ELISA效价为1∶256~1∶512,腹水效价为1∶12800~1∶25600,间接ELISA显示其中4株McAb1F3、2H5、4A1、4H9能与恶性疟原虫抗原发生特异性结合,而只有1F3、4A1、4H9在Westernblot试验中识别恶性疟原虫Mr约35000的虫源蛋白。结论获得了能稳定分泌高特异性抗EBA175McAb的杂交瘤细胞。
Objective To prepare a monoclonal antibody against Plasmodium falciparum EBA-175 (McAb), which laid the foundation for the diagnosis and vaccine research of Plasmodium falciparum. Methods BALB / c mice were immunized with the recombinant antigen of Plasmodium falciparum EBA-175 (F2 of region II). McAbs were prepared by hybridoma technique. The hybridoma cell lines secreting high titer McAbs were screened by indirect ELISA. The immunoglobulin subunits Class and its potency, ELISA, Westernblot test analysis of its specificity. Results Six hybridoma cell lines, 1F3, 2E8, 2H5, 4A1, 4C3, 4H9, and 6 McAbs stably secreting anti-recombinant EBA-175McAb were screened and identified as 5 subtypes of IgG1, 1 IgG2a and 6 McAbs The supernatant ELISA titer was 1:256 ~ 1: 512, the ascites titer was 1:12800 ~ 1:25600, indirect ELISA showed that 4 McAb1F3, 2H5, 4A1, 4H9 were specific to Plasmodium falciparum antigen Whereas only 1F3, 4A1, 4H9 recognized about 35,000 Mr protein from P. falciparum in the Western blotting assay. Conclusion The hybridoma cells that can stably secrete high specific anti-EBA175McAb were obtained.