Production Of Monoclonal Antibodies To Rice Stripe Virus And Application In Virus Detection

Rice stripe disease is caused by Rice stripe virus (RSV), the disease epidemic is common in some parts of Jiangsu province, and has become a major disease to rice production in the province. The most effective ways for controlling the disease are cultivation of resistant varieties and killing the insect vector. The proportion of the viruliferous Laodelphax striatellus is the most important factor for the epidemic of the disease, so a rapid and sensitive method is necessary for detection of RSV in Laodelphax striatellus. Monoclonal antibodies (MAbs) of RSV were produced and detection methods are established using the MAbs.Four hybridoma cell lines secreting MAbs against RSV were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized by the RSV particles. The titres of ascitic fluids of four MAbs ranged from 1:80000 to 1:5120000 when tested by indirect ELISA. Indirect ELISA was established with the produced MAbs for RSV detection, and the four MAbs could successfully detect RSV in plant sap with 1:2560 dilution. The MAbs didn't cross-react with other tested plant viruses. The result of Western-blot showed that the four MAbs could react with the 35 kD RSV coat protein specifically. Indirect ELISA was then used for detection of RSV in Laodelphax striatellus Fallen. The proportion of the viruliferous Laodelphax striatellus in Jiangsu province ranged from 12.5% to 41.5%.Monoclonal antibodies 3B9, 2H2 and 2E5 were purified by saturated (NH4)2SO4 The content of IgG was 6. 52 mg/ml, 10.25 mg/ml and 9.64 mg/ml, respectively. The horseradish peroxidase-linked antibodies HRP-3B9, HRP-2H2 and HRP-2E5 were produced by NaIO4 method. The titres of the three enzyme-linked antibodies were 1:25600, 1:1600 and 1:3200, respectively. In direct ELISA, the suitable diluted times of the three enzyme-linked antibodies were 1:5000, 1:1000 and 1:1500, respectively. Enzyme-linked antibody HRP-3B9 was selected to detect RSV in Laodelphax striatellus Fallen using direct dot immunobinding assay (DIBA) and indirect DIBA. Detection results showed that DIBA could detect RSV in the insect and direct DIBA was more sensitive than indirect DIBA. The direct DIBA was then used to detect the virus in field collected Laodelphax striatellus Fallen.