建兰花叶病毒单克隆抗体的制备及检测应用

Production of monoclonal antibodies to Cymbidium mosaic virus and application in Orchids virus detection

摘要:

用建兰花叶病毒(Cymbidium mosaic virus,CymMV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得3株能稳定传代并分泌抗CymMV单克隆抗体(McAb)的杂交瘤细胞(2C6、5B7和12G9),分别制备它们的单抗腹水。其中5B7和12G92株单克隆抗体腹水间接ELISA效价达10-6,3株单抗的抗体类型及亚类均为IgG1,轻链均为κ链。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)检测CymMV的方法。蝴蝶兰病叶作1∶10240倍稀释、提纯CymMV病毒浓度为4.87ng/mL(每孔的病毒绝对量为0.487ng)时,该方法仍能检测到病毒。利用ACP-ELISA方法检测了田间样品,发现CymMV在兰花上发病很普遍。

关键词:建兰花叶病毒; 单克隆抗体; ACP-ELISA

Abstract:

Three hybridoma cell lines, 2C6, 5B7 and 12G9, secreting monoclonal antibodies (McAbs) against Cymbidium mosaic virus (CymMV) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/C immunized by the CymMV particles. The three McAbs could specifically react with CymMV. The titres of ascitic fluids of two McAbs are up to 10-6 in I-ELISA. Isotypes and subclasses of the the three McAbs belong to IgG1. Isotypes of light strains of the three McAbs all belong to κ. They were used in antigen-coated plate (ACP) -ELISA for CymMV detection, and ACP-ELISA could successfully detect 0.487ng of purified CymMV or virus in plant sap diluted 1∶10240. The presence of CymMV in field Orchids tissues was investigated with ACP-ELISA.

Key words:Cymbidium mosaic virus; monoclonal antibodies; ACP-ELISA