Three highly sensitive monoclonal antibody-based serological assays for the detection of tomato mottle mosaic virus

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.