摘要
为建立简便、有效、低成本小麦黄花叶病毒(WYMV)的检测方法,用提纯的WYMV免疫BALB/c小鼠后,利用杂交瘤细胞技术经细胞融合、筛选和克隆,共获得2D4、2D3和6F4 3株能稳定传代并分泌抗WYMV单克隆抗体的杂交瘤细胞,并分别注射小鼠腹腔制备其单抗腹水。3株单抗腹水的间接ELISA效价为10^(-6)~10^(-7),抗体类型及亚类均为IgG1、κ链。特异性检测结果表明6F4和2D4这2株单抗仅与WYMV的32 kD外壳蛋白有特异性免疫反应。利用效价最高的2D4单抗建立了检测WYMV的抗原包被ELISA方法(ACP-ELISA),其灵敏度分析结果表明,当病叶以1∶25 600(g/ml)倍稀释时仍能检测到病毒,特异性分析结果表明可有效区分小麦易感染的中国小麦花叶病毒(CWMV)。田间样品检测结果表明该方法可准确、可靠地用于小麦WYMV的大规模检测。
To develop a simple,effective and low-cost method for detecting wheat yellow mosaic virus( WYMV),the purified WYMV virus particles were used as an immunogen,after fusioned,selected and cloned,three hybridoma cell lines( 2D4,2D3 and 6F4) secreting monoclonal antibodies( MAbs) against WYMV were produced by fusing mouse myeloma cells with spleen cells from BALB/c,and injected intraperitoneally into BALB/c mice to produce ascetic fluids. The titers of three MAbs in ascites determined by an indirect-ELISA ranged from 10^(-6) to 10^(-7). Isotypes and subclasses of three MAbs belonged to IgG1,κlight chain. 6F4 and 2D4 MAbs could specifically react with coat protein( 32 kD) of WYMV.Based on the most sensitive 2D4 MAb,an antigen-coated plate enzyme-linked immunosorbent assay( ACP-ELISA) for detecting WYMV was established. Sensitivity analysis results showed that the virus could be detected when the diseased leaves were diluted with 1 ∶ 25 600( g/ml) times,specificity analysis results showed that the method could be used to distinguish WYMV and Chinese wheat mosaic virus( CWMV). The field sample detection results suggested that the ACP-ELISA could accurately and reliably detect WYMV in large-scale wheat samples.
出处
《江苏农业学报》
CSCD
北大核心
2018年第1期34-40,共7页
Jiangsu Journal of Agricultural Sciences
基金
国家转基因重大专项(2016ZX08002)
