Three highly sensitive monoclonal antibody-based serological assays for the detection of tomato mottle mosaic virus
Three highly sensitive monoclonal antibody-based serological assays for the detection of tomato mottle mosaic virus
复制标题
三种基于单克隆抗体的高灵敏度血清学检测方法检测番茄斑驳花叶病毒
DOI：
10.1186/s42483-021-00100-2
复制
发表时间：
2021-09-18
影响因子：
3.4
通讯作者：
Wu, Jianxiang
中科院分区：
文献类型：
--
作者：
Li, Xue;Guo, Liqian;Wu, Jianxiang
近年来，番茄斑驳花叶病毒（Tomato mottle mosaic virus，ToMMV）已成为影响我国云南、海南、山东等地茄科作物生产的重要病毒病原之一，常造成严重减产。为了给农民和蔬菜行业提供可靠、简便的ToMMV检测方法，我们用纯化的ToMMV免疫BALB/c小鼠，获得6株杂交瘤细胞株（即，2D 6、9 C12、26 A10、3A 4、23 A4和17 B11），其通过杂交瘤技术分泌抗ToMMV单克隆抗体（MAb）。以这些单克隆抗体为检测抗体，建立了ToMMV的抗原包被平板酶联免疫吸附试验（ACP-ELISA）、斑点酶联免疫吸附试验（dot-ELISA）和组织印迹酶联免疫吸附试验（tissue print-ELISA）。我们的试验结果表明，这三种新开发的血清学方法可以特异性地检测植物样品中ToMMV的感染，但不能检测烟草花叶病毒、番茄花叶病毒、黄瓜绿色斑驳花叶病毒和黄瓜花叶病毒。敏感性分析进一步表明，ACP-ELISA和dot-ELISA可分别用于检测稀释度为1：81，920和1：40，960（重量/体积，g/mL）的植物粗提物中ToMMV的感染。令人惊讶的是，所开发的dot-ELISA的检测限比传统的RT-PCR高26倍。使用现场采集的植物样品，我们已经证明，这三种新的血清学方法是准确的，易于使用的大规模检测ToMMV在田间。
In recent years, tomato mottle mosaic virus (ToMMV) has become one of the most important viral pathogens affecting solanaceous crop production in Yunnan, Hainan, and Shandong provinces of China, often causing huge yield reductions. To provide farmers and vegetable industry with reliable and easy-to-use ToMMV detection methods, we immunized BALB/c mice with purified ToMMV and obtained six hybridoma cell lines (i.e., 2D6, 9C12, 26A10, 3A4, 23A4 and 17B11) that secrete anti-ToMMV monoclonal antibodies (MAbs) through the hybridoma technology. Using these MAbs as the detection antibody, we developed three serological assays: antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and tissue print enzyme-linked immunosorbent assay (tissue print-ELISA) for ToMMV detection. Our test results showed that these three newly developed serological methods can be used to specifically detect ToMMV infection in plant samples, but not tobacco mosaic virus, tomato mosaic virus, cucumber green mottle mosaic virus and cucumber mosaic virus. Sensitivity analyses further showed that ACP-ELISA and dot-ELISA can be used to detect ToMMV infection in plant crude extracts diluted at 1:81,920 and 1:40,960 (weight/volume, g/mL), respectively. Surprisingly, the detection limit of the developed dot-ELISA was 26 times higher than that of traditional RT-PCR. Using field-collected plant samples, we have demonstrated that these three new serological methods are accurate and easy-to-use for large-scale detection of ToMMV in fields.