Production And Application Of Monoclonal Antibodies To Macrobrachium Rosenbergii Nodavirus

Whitish muscle diseases of Macrobrachium rosenbergii was a new epidemic disease in the larvae and post-larvae of M. Rosenbergii in recent years. It first occurred in Guangdong province in 1996 in China, and then broke out in 2000. The clinical sign of the disease is a whitish or opacity appearance of the muscles in abdomen. Mortalities may reach more than 60%, sometime even reach 100%.The pathogen of the disease was confirmed as Macrobrachium rosenbergii Nodavirus(MrNV). MrNV is an icosahedron, non-enveloped viral particle sized from 25~26nm. The genome of MrNV is composed of two linear single-stranded positive RNAs sized 3.0kb and 1.3kb respectively. MrNV is the first nodavirus founded in crustacean.Mouse myeloma cells (SP2/0) were fused with spleen cells from BALB/c immunized with purified MrNV. Positive colonies were selected by indirect ELISA. After 2-3 times cloning with limited dilution, twelve hybridoma cell lines secreting monoclonal antibodies(Mabs) against MrNV were used for ascitis production. The titres of ascitic fluids of twelve Mabs ranged from 1 : 10~5 to 1 : 10~6 by indirest ELISA. Isotypes of the tweleve Mabs were tested by SBA clonotyping system, the result showed that six Mabs were characterized as IgG1, four Mabs were IgG2a and two Mabs were IgG2b. The twelve Mabs didn't cross react with other main viruses of shrimp, WSSV and TSV. 2B5 can react with 43kD coat protein of MrNV by Western blot.The method of three antibodies sandwich-ELISA(TAS-ELISA) was established by using Mab 2B5 and rabbit antibody angainst MrNV. The optimun condition of TAS-ELISA: rabbit antibody used for plate precoating was 1 μ g/mL, work concentration of Mab 2B5 was 1 : 50, 000. The sensitivity for detection MrNV by TAS-ELISA from the puried MrNV was 0.98ng. The positive detection rate as well as coincident rate were compared with different methods and showed that TAS-ELISA was better than indirect ELISA and RNA characterization with electrophoresis, when the diseased and suspicious prawn samples were used for analysis. Besides, a rapidTAS-ELISA was developed with shorter incubation time at lower temperature, whichmaking TAS-ELISAmore practical in field detection.