细胞表面荧光免疫吸附法在猪流行性腹泻病毒N蛋白单克隆抗体杂交瘤筛选中的应用

Application of cell surface-fluorescence immunosorbent assay in rapid screening of a hybridoma secreting monoclonal antibody against PEDV N protein

为验证细胞表面荧光免疫吸附法(CSFIA)筛选高活性杂交瘤细胞的效果,本研究以原核表达的猪流行性腹泻病毒(PEDV)重组N蛋白为免疫原,免疫6周龄~8周龄的BALB/c雌性小鼠,取其脾细胞与SP2/0细胞融合,分别采用CSFIA和间接ELISA方法筛选杂交瘤细胞,并检测目的杂交瘤细胞分泌的单克隆抗体(MAb)效价。结果显示,CSFIA筛选的MAb 1B1比间接ELISA筛选的MAb 3D3抗体效价高10倍,并且通过观察荧光强度能够实现杂交瘤细胞的一次性筛选,无需多次亚克隆,周期缩短约30 d。本研究表明CSFIA在筛选杂交瘤细胞中具有简单、直观和高效的优点,是一种值得推广的快速制备MAb的新方法。

To verify the effect of cell surface-fluorescence immunosorbent assay(CSFIA)on screening the highly active hybridoma cells,6-8 week-old female BALB/c mice were immunized with recombinant porcine epidemic diarrhea virus(PEDV)N protein,and then spleen cells isolated from mice were fused with SP2/0 cells to generate hybridomas.The hybridomas secreting MAbs against PEDV N protein were screened by indirect ELISA and cell surface-fluorescence immunosorbent assay(CSFIA),respectively.The titer of MAbs secreted by each hybridoma was determined.Using these methods,two strains of hybridoma cell lines secreting MAbs against PEDV N protein,designated as 3 D3 and 1 B1,respectively,were obtained and characterized.The results showed that the titer of 1 B1 produced by CSFIA was 10 times higher than that of 3 D3,and the preparation time was shortened by about 30 days on account of the direct selection of hybridoma through the fluorescence intensity without the need of multiple subcloning procedures.This study indicates that CSFIA is a powerful method to screen the hybridomas secreting higher titer MAb in a rapid one-step process,which would be valuable alternative for a rapid production of MAbs.