Abstract: 【Objective】The aim of this study is to prepare monoclonal antibodies (MAbs) against Watermelon mosaic virus (WMV) and develop effective serological assays for rapid and reliable virus detection, and to provide technology and materiel for diagnosis and detection, forecast and early warning and establishment of a scientific prevention and control system of the WMV disease.【Method】Using the purified WMV particles as an immunogen, hybridoma lines secreting MAbs specific for WMV were obtained via cell fusion, cell culture, antibody detection and cell cloning. The hybridomas were injected intraperitoneally into BALB/c mice to produce MAb-containing ascitic fluids. Based on the prepared MAbs, five detection assays, ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot-ELISA and IC-RT-PCR were developed for accurately, sensitively and specifically detecting WMV in field plant samples. Besides, a dot-ELISA for specifically detecting WMV in individual viruliferous aphid was established.【Result】Three hybridoma lines (2C8, 15A8 and 16C12) steadily secreting MAbs specific for WMV and their MAb-containing ascitic fluids were produced. The titers of ascitic fluids of MAbs were up to 10-6 by indirect-ELISA. All these MAbs belong to IgG1 isotype, κ light chain. Western blot analyses indicated that all these three MAbs could specifically react with the coat protein of WMV. The ACP-ELISA, DAS-ELISA, dot-ELISA and IC-RT-PCR could detect WMV in infected plant crude extracts diluted up to 1﹕163 840, 1﹕327 680, 1﹕5 120 and 1﹕1 310 720 (w/v, g/mL), respectively. And the specificity analyses demonstrated that the developed ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot-ELISA and IC-RT-PCR had strongly positive immune reactions with WMV-infected plant tissues, but had negative reactions with healthy, ZYMV-, CMV-, CGMMV-infected cucurbitaceous plant tissues or PVY-infected tobacco plant tissues. Besides, the developed dot-ELISA for detecting vector sample had a strongly positive immune reaction with individual viruliferous aphid, and negative reactions with non-viruliferous aphids. A total of 275 cucurbitaceous plant samples showing virus-like symptoms from Zhejiang, Jiangsu, Shandong and Hainan provinces in China were screened for the presence of WMV using the developed assays, and the detection results showed that 187 of the 275 plant samples were infected by WMV and the incidence rate was up to 68%, demonstrating that WMV is prevalent in field cucurbitaceous plants in China. And the detection results of serological assays were in agreement with those of RT-PCR. The sequences of PCR-amplified products were sequenced and compared with the WMV CP sequences. The results indicated that the nucleotide sequences of the PCR-amplified products were WMV CP gene segment, demonstrating that the positive samples tested by serological assays were really infected with WMV.【Conclusion】Three specific and sensitive MAbs against WMV and the five developed assays based on prepared MAbs in this study could accurately, sensitively and reliably detect WMV in field plant or vector samples, which would provide technology and materiel for rapid detection and diagnoses of field large-scale samples, forecast and early warning, scientific prevention and control of WMV disease in China.

Key words: Watermelon mosaic virus (WMV), monoclonal antibody, ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot- ELISA, IC-RT-PCR