香石竹斑驳病毒单克隆抗体的制备及检测应用

 用香石竹斑驳病毒(CarMV)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5株能稳定传代且分泌抗CarMV单克隆抗体(MAb)的杂交瘤细胞,并分别制备它们的单克隆抗体腹水。5株单克隆抗体腹水间接ELISA效价达10^-6,其中3G1、1B9、2A9和2F8的抗体类型及亚类均为IgG1,而2F₂为IgG3。Western-blot分析表明,5株单克隆抗体均与CarMV 38 kD的外壳蛋白亚基有特异性反应。利用2A9单抗建立的抗原包被的间接ELISA (ACP-ELISA)检测CarMV方法,病叶1:800倍稀释、提纯CarMV病毒浓度为1 ng/mL (绝对检测量为0.1 ng)时仍能检测到病毒。利用ACP-ELISA对田间香石竹样品的检测表明,CarMV在香石竹上发病很普遍。

Production of monoclonal antibodies to Carnation mottle virus and its application in virus detection

Five hybridoma cell lines secreting monoclonal antibodies (MAbs) against Carnation mottle virus (CarMV) were produced by fusing mouse myeloma cells (Sp2/0) with spleen cells from BALB/c mice immunized by the CarMV particles. The five MAbs could specifically react with CarMV. The titres of ascitic (fluids) of the five MAbs are 10~(-6) in indirect antigen-coated plate (ACP)- ELISA. Isotypes and subclasses of 3G1,1B9,2A9 and 2F8 belong to IgG1 while that of 2F2 belong to IgG3. Western-blot analysis showed that all the five MAbs can react specifically with the 38 kD coat protein subunit of CarMV. The subclass 2A9 was used in ACP-ELISA for detection of CarMV. ACP-ELISA could successfully detect 0.1 ng purified CarMV or virus in plant sap diluted 800 fold. The presence of CarMV in field Dianthus caryophyllus tissues was investigated with ACP-ELISA and the detection results showed that CarMV is common in field samples of Dianthus caryophyllus.