title: Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein.
abstract: The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol µl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.
authors.affiliation: Department of Biology, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand.
authors.forename: Pradit
authors.initials: P
authors.lastname: Wangman
authors.forename: Saengchan
authors.initials: S
authors.lastname: Senapin
authors.forename: Parin
authors.initials: P
authors.lastname: Chaivisuthangkura
authors.forename: Siwaporn
authors.initials: S
authors.lastname: Longyant
authors.forename: Sombat
authors.initials: S
authors.lastname: Rukpratanporn
authors.forename: Paisarn
authors.initials: P
authors.lastname: Sithigorngul
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000276:immunology*; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005393:Fish Diseases / Q000175:diagnosis / Q000821:virology*; D051379:Mice; D030381:Nodaviridae / Q000276:immunology*; D033541:Palaemonidae / Q000821:virology*; D012327:RNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D011934:Reagent Strips; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; D011934:Reagent Strips; D011994:Recombinant Proteins