抗华支睾吸虫成虫抗原McAb制备及功能鉴定的研究

Study on the development and function of a monoclonal antibody to clonorchis sinensis

目的利用杂交瘤技术建立分泌抗华支睾吸虫成虫抗原的单克隆抗体杂交瘤细胞株并对其分泌抗体的免疫学活性进行初步鉴定。方法以华支睾吸虫成虫抗原免疫BALB／c小鼠，取其脾细胞与Sp2／0骨髓瘤细胞进行融合，筛选分泌高滴度单克隆抗体的杂交瘤细胞株，采用动物体内诱生的方法制备单克隆抗体并对其纯化，测定所制备单抗效价和其免疫球蛋白亚类，检测单抗与猪囊尾蚴、弓形虫、疟原虫和日本血吸虫抗原的交叉反应，观测单抗对虫体的识别部位和其与感染小鼠肝脏组织中抗原的结合情况。结果获得1株分泌高滴度抗华支睾吸虫成虫抗原的单克隆抗体杂交瘤细胞株，分泌的单抗命名为D10。其效价达1：200000以上，纯度达90％以上，亚类鉴定为IgGl类；单抗株D10与猪囊尾蚴、弓形虫、疟原虫和日本血吸虫抗原无交叉反应；免疫荧光法见其与华支睾吸虫成虫表皮及虫卵结合；免疫组化显示单抗株D10与病变肝脏组织中汇管区及周围间质中的抗原结合明显。结论制备的单抗株D10具有高效价和高特异性，为华支睾吸虫病的研究及华支睾吸虫病诊断抗体的研制奠定了基础。

Objective To establish hybridoma cell lines secreting monoelonal andibody （McAb） against antigens of adult Clonorchis sinesis and to identify its immunological activity of secreating antibody. Methods The BALB/c mice were immunized with antigens of adult clonorchis sinesis and the spleen cells of immunized mice were fused with myloma cell line SP2/0. Then the bybridoma cell lines secreting high titer of McAbs were screened. The Ig subtypes and titer of the McAbs were assayed with ELISA. Hybridoma cells were injected into the abdominal cavity of BALB/C mice to pre- pare and purify the ascites of McAb. The purity and the titer of the ascites were detected. The cross reactions between the McAb and antigens of Cysticercosis, Toxoplasma, Plasmodium, Schistosoma japonicum were identified. Examined with IF- AT to identify the recognition parts of the polypide with McAb. Meanwhile, the localization of Ag was determined with McAb by immunohistochemistry technique in the liver of infection. Results One bybridoma cell line secreting high titer of McAbs against antigens of adult Clonorchis sinensis were established. The McAb was named D10. Its subclass was found to be IgG1. It had only one eluting peak under the observation of FPLC and the titer of the McAb D10 was 1：200 000. The McAb had no cross reactions with antigens of Cysticercosis, Toxoplasma, Plasmodium, Schistosoma japonicum. IFAT showed that the McAb D10 identified the epidermis and egg of the polypide. The Ag in portal area and interstitium surrounding of the pathological changes of the liver tissues was observed obviously with McAb by immunohis- tochemistry technique. Conclusion One of the McAb against adult Clonorchis sinensis antigen was successfully achieved, named D10. The D10 has advantages in high purity and immunologic competence, which lay the foundation for the research of Clonorchis sinensis and the trituration of diagnosis antibody.