摘要
目的探讨高尔基体蛋白73(GP73)鼠源单克隆抗体的制备方法并进行鉴定。方法用GP73重组抗原蛋白免疫5只小鼠获得血清抗体,免疫小鼠脾细胞与sp2/0骨髓瘤细胞融合,经过筛选后选择GP73抗体阳性杂交瘤细胞进行克隆化培养,通过腹水制备获得高特异性和高效价的GP73单克隆抗体(单抗)。结果成功筛选出11株能稳定分泌特异性GP73单抗的杂交瘤细胞株,经多次复苏传代仍能稳定分泌特异性强、效价高的抗体。11株单抗均能结合天然状态下的GP73蛋白,与无关蛋白均无交叉反应,提示有较强的特异性。应用筛选出的配对GP73单抗可检测出浓度为1ng/ml的GP73基因工程表达抗原。结论制备的GP73杂交瘤细胞株分泌的抗体对GP73蛋白具有特异亲和性,并且有较高效价,为GP73蛋白诊断试剂的研制提供了关键技术基础。
Objective To establish hybridoma cell line secreting monoclonal antibody （McAb） by Golgi protein 73 （GP73） and identify the McAb. Methods Five Balb/c mice were immunized with GP73. The spleen cells of immunized mice were fused with sp2/O cells, screened and cloned. Thus ascites with McAb was prepared. The specificity of GP73 McAb was identified by ELISA and cell immunofiuorescence. Results Eleven hybridoma cell lines which could stably secrete specific McAb against GP73 were established with perfect specificity and higher efficiency. After several times of resuscitation and regeneration, the ability of secreting McAb by these eleven cell lines still kept on their specific quality. The specific identifications of GP73 MeAb showed positive reaction very well. The concentration of antigen of GP73 expression of gene engineering was 1 ng/ml by using paired GP73 monoclonal antibody. Conclusion The GP73 McAb secreted by the eleven hybridoma cell lines shows perfect specificity and strong affinity with GP73, which provides the key technical foundation on development of GP73 diagnostic reagent.
出处
《北京医学》
CAS
2013年第6期440-442,I0003,共4页
Beijing Medical Journal
基金
北京市肝病研究所2012年度双十计划项目(NO.Z121107002812113)
