Enhanced white spot syndrome virus (WSSV) detection sensitivity using monoclonal antibody specific to heterologously expressed VP19 envelope protein

Abstract

The gene encoding the VP19 envelope protein of white spot syndrome virus (WSSV) was cloned into pMAL-C2 expression vector and transformed into the BL21 Escherichia coli strain. After induction, recombinant maltose binding protein (MBP)-VP19 fusion protein was produced, purified and electroeluted before use for immunization in Swiss mice for monoclonal antibody (MAb) production. MAbs specific to VP19 can be used to detect natural WSSV infection in Penaeus vannamei by dot blotting, western blotting and immunohistochemistry without cross-reaction to other shrimp tissues or other common shrimp viruses, including Taura syndrome virus (TSV), yellow head virus (YHV), Penaeus monodon nucleopolyhedrovirus (PemoNPV) formerly called monodon baculovirus (MBV) and P. monodon densovirus (PmDNV) previously called hepatopancreatic parvovirus (HPV). The detection sensitivity of the VP19-specific W25-8D MAb generated in this study was approximately 1.2 fmol/spot of purified recombinant MBP-VP19 protein as determined by dot blotting while that of a VP28-specific W29 MAb obtained from a previous study was approximately 5 fmol/spot. Combining MAbs specific for VP19 and VP28 resulted in two-fold higher sensitivity than use of either MAb alone. However, the sensitivity of the combined MAbs was 25,000 times lower than that of one-step PCR. Immunohistochemical analysis using MAbs specific to VP19 in WSSV-infected gill tissues and appendages demonstrated intense staining patterns in both the nucleus and cytoplasm compared to MAb specific to VP28. In conclusion, combination of VP19- and VP28-specific MAbs could confirm and enhance the sensitivity of WSSV detection in shrimp in various types of antibody-based assays. © 2009 Elsevier B.V. All rights reserved.