Characterization and application of monoclonal antibodies against white spot syndrome virus

Abstract

Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north‐eastern Taiwan. By sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG1 class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non‐target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies.

Monoclonal antibodies developed for sensitive detection and comparison of white spot syndrome virus isolates in India

“…Among the various WSSV proteins, the 28 kDa protein has been found to be dominant and highly immunogenic (Hameed et al 1998, Poulos et al 2001). However, an MAb reacting only with the 19 kDa protein has also been produced (Shih et al 2001). The 4 MAbs differed in reactivity with the viral proteins, as indicated by the intensity of colour in the Western blot and immunodot tests.…”

“…Antigen analysis of the virus has been carried out using polyclonal rabbit antisera, also used for immunodiagnosis (Nadala et al 1997, Hameed et al 1998. Recently, monoclonal antibodies against the Chinese isolate of WSSV have also been produced and characterised (Yu et al 1995, Zhan et al 1999, Poulos et al 2001, Shih et al 2001.…”

“…MAb-based tests such as immunofluorescence (Zhan et al 1999, Shih et al 2001, immunoperoxidase and whole-mount tissue assays (Poulos et al 2001) have been developed for detection of the Chinese isolate of WSSV. However, such tests are not ideally suited for field level screening by farmers.…”

“…This would require application of less-sensitive PCR (1-step) or an alternative screening procedure of similar sensitivity. In this context, we attempted to develop a simple and sensitive monoclonal antibody (MAb)-based assay that would be suitable for fieldlevel screening of shrimp for WSSV.MAb-based tests such as immunofluorescence (Zhan et al 1999, Shih et al 2001), immunoperoxidase and whole-mount tissue assays (Poulos et al 2001) have been developed for detection of the Chinese isolate of WSSV. However, such tests are not ideally suited for field level screening by farmers.…”

Evaluation of an immunodot test to manage white spot syndrome virus (WSSV) during cultivation of the giant tiger shrimp Penaeus monodon

“…Monoclonal antibody (MAb) based assays developed for detection of WSSV include immunoperoxidase and whole-mount tissue assays (Poulos et al 2001), immunofluorescence assay (Shih et al 2001), immunodot assay (Anil et al 2002), dot -immunogold filtration (DIGF) assay ), a single-step immunochromatographic test , and a Shrimple ® test strip (EnBioTec Laboratories). Among the various DNA-and antibody-based assays for WSSV, PCR is very sensitive and is used widely for WSSV screening in shrimp hatcheries and grow-out ponds.…”