DAO 65:85-89 (2005)  -  doi:10.3354/dao065085

Generation of monoclonal antibodies specific to hepatopancreatic parvovirus (HPV) from Penaeus monodon

Sombat Rukpratanporn1, Wasana Sukhumsirichart2, Parin Chaivisuthangkura3, Siwaporn Longyant3, Weerawan Sithigorngul3, Piamsak Menasveta1, Paisarn Sithigorngul3,*

1Marine Biotechnology Research Unit at Chulalongkorn University, National Center for Genetic Engineeringand Biotechnology (BIOTEC), Bangkok 10330, Thailand
2Department of Biochemistry, and 3Department of Biology, Srinakharinwirot University, Bangkok 10110, Thailand
*Corresponding author. Email: paisarn@swu.ac.th

ABSTRACT: Hepatopancreatic parvovirus (HPV) was isolated from the hepatopancreas (HP) of slow growth Penaeus monodon by urografin gradient centrifugation. The presence of HPV in the fraction was monitored by PCR and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Only 1 major 54 kDa protein band was observed in the strong PCR-positive fractions used to immunize mice for monoclonal antibody production. After cell fusion, the first step in selecting specific antibodies was performed by dot-blot assay with purified HPV viral particles. The second screening step was carried out using Western blots of purified HPV proteins and immunohistochemistry of HPV-infected HP tissue. Four monoclonal antibodies were isolated; these bound to the 54 kDa protein in Western blots and to intranuclear inclusion bodies in tubule epithelial cells of HPV-infected prawn tissue by immunohistochemistry. None of the antibodies showed cross-reactivity either to uninfected shrimp tissue or to other shrimp viruses tested. These reagents have potential for use in developing a highly sensitive immunoassay such as sandwich ELISA or a convenient kit for detection of HPV infection.


KEY WORDS: Dot-blot · Hepatopancreatic parvovirus · HPV · Immunohistochemistry · Monoclonal antibody · Penaeus monodon


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