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| Development of Monoclonal Antibodies Against Chinese wheat mosaic virus (CWMV) and Their Application |
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Abstract Chinese wheat mosaic disease, which is caused by Chinese wheat mosaic virus (CWMV), leads to severe yield and huge economic losses on wheat (Triticum aestivum). The purpose of this study was to develop serological methods for detecting CWMV using prepared monoclonal antibodies (MAbs) against CWMV, and to provide technical support for the diagnosis and scientific prevention and control of CWMV in fields. Using the purified CWMV virus particles as an immunogen, 4 hybridoma lines (5H5, 7C2, 9A7 and 12E5) secreting MAbs against CWMV were obtained via cell fusion, cell culture, antibody detection and cell cloning, and injected intraperitoneally into BALB/c mice (Mus musculus) to produce ascitic fluids. The titers of all 4 MAbs in ascites determined by an indirect-ELISA were up to 10-7. Isotypes and subclasses of all 4 MAbs belonged to IgG1, κ light chain. Western blot analysis indicated that all 4 MAbs could specifically react with the coat protein of CWMV. Using the MAbs, antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA) and dot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting CWMV were established, respectively. Specificity analysis of the ACP-ELISA and the dot-ELISA indicated that both 2 serological detection methods had positive reactions of detection with CWMV infected wheat plant tissue crude extracts and negative reactions of detection with wheat or barley (Hordeum vulgare) plant infected by Wheat yellow mosaic virus (WYMV) or Barley yellow mosaic virus (BaYMV) and healthy wheat plant. This result suggested that 2 serological detection methods could specifically detect CWMV in wheat plants. Sensitivity analysis showed that the sensitivity of the ACP-ELISA based on the MAbs (5H5 and 12E5) for detecting CWMV in infected wheat tissue could reach 1∶81 920(W/V, g/mL), while the detection sensitivity of ACP-ELISA based on the MAbs (7C2 and 9A7) was up to 1∶40 960 (W/V, g/mL). The sensitivity of the dot-ELISA based on the MAbs (5H5 and 12E5) for detecting CWMV in infected wheat tissue could reach 1∶2 560 (W/V, g/mL), while the detection sensitivity of the dot-ELISA based on the MAbs (7C2 and 9A7) was up to 1∶1 280 (W/V, g/mL). The field sample detection results suggested that 2 serological detection methods could accurately and reliably detect CWMV in field wheat samples, and furtherly confirmed that CWMV was prevalent in Jiangsu and Shandong Provinces. The anti-CWMV MAbs and the developed serological detection methods provide material and technology for diagnosis, forecast and scientific prevention and control of CWMV.
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Received: 19 December 2014
Published: 13 April 2015
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