Production Of Monoclonal Antibodies Against Apple Stem Grooving Virus And Their Application In The Virus Detection

Apple stem grooving virus (ASGV) is one of the most common viruses infecting pear and apple, and usually decreases the fruit production and quality, and causes a lot of economic loss of pear and apple worldwide. In order to develop a high-speed and sensitive detection method, monoclonal antibodies (MAbs) were raised against ASGV vrions and used to detect the virus in different pear and apple samples. The study provides effective serological reagents for the ASGV detection. The obtained results are listed as followings:1. In vitro pear sample "Huanghua" infected by ASGV as identified by RT-PCR was used as virus source. Chenopodium quinoa and Nicotiana occidentalis were inoculated with extracts from "Huanghua" leaves. The symptoms of systemic chlorotic mottling and chlorotic spots on C. quinoa and systemic chloroctic spots and necrotic spots on N. occidentalis were observed at7day post inoculation. Then, C. quinoa plants were used as propagation host plants and inoculated with extracts from ASGV infected C. quinoa.2. ASGV virions were purified from100g leaves of ASGV infected C. quinoa. The concentration of obtained1mL viral extracts was4.29mg/mL. The presence of ASGV in the extract were confirmed by SDS-PAGE, Western blot and RT-PCR.3. The spleen cells from mouse BALB/C immunized with the ASGV virions were fused with myeloma cells (SP2/0). Two hybridoma cell lines specifically secreting MAbs against prokaryotic expressing recombinant coat proteins (rCPs) of ASGV were screened. The titres of ascetic fluids of two MAbs are1:25600as tested by indirect ELISA. The two Mabs showed to have high specificity to ASGV coat protein as indicated by Western blot assay. Prokaryotic expressing rCPs of ASGV were used to test the sensitivity of two Mabs by ID-ELISA. The MAbs2C3and1F10could detect minimal rCPs of28ng/mL and2.8ng/mL, respectively.4. The presence of ASGV in49pear and apple samples was investigated by TAS-ELISA using the mixture of MAbs2C3and1F10. The result showed29samples were positive to ASGV, which was compariable to the RT-PCR detection results, except for one sample to be positive in RT-PCR and negative in TAS-ELISA test.