Journal of Virological Methods 133 (2006) 180–184 Development of a polyclonal antibody specific to VP19 envelope protein of white spot syndrome virus (WSSV) using a recombinant protein preparation Parin Chaivisuthangkura a , Phiromsak Phattanapaijitkul b , Nitaya Thammapalerd b , Sombat Rukpratanporn c , Siwaporn Longyant a , Weerawan Sithigorngul a , Paisarn Sithigorngul a,∗ a Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand b Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand c Center of Excellence for Marine Biotechnology at Chulalongkorn University, National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok 10330, Thailand Received 4 July 2005; received in revised form 8 November 2005; accepted 8 November 2005 Abstract The VP19 gene encoding a structural envelope protein of white spot syndrome virus was cloned into an expression vector and introduced into E. coli. The objective was to produce a recombinant VP19 structural protein. After induction, the recombinant VP19 protein (rVP19) was produced, purified by SDS-PAGE and used for immunization of Swiss mice for polyclonal antibody production. The mouse anti rVP19 antiserum had specific immunoreactivity to the viral antigen in WSSV infected Penaeus monodon as verified by immunohistochemistry and Western blot. The production of monoclonal antibodies against this rVP19 may be useful in order to combine with anti-VP28 monoclonal antibodies for enhancing the sensitivity of various WSSV serological assays. © 2006 Elsevier B.V. All rights reserved. Keywords: Immunohistochemistry; Penaeus monodon; Polyclonal antibody; VP19; Western blot; WSSV 1. Introduction White spot syndrome virus (WSSV) is one of the most viru- lent pathogen that causes major losses in shrimp farming world- wide. WSSV contains five major structural proteins, VP28 and VP19 are located in the envelope and VP26, VP24 and VP15 are located in the nucleocapsid (Van Hulten et al., 2001). Fur- ther characterization of WSSV revealed that VP19 contained two putative transmembrane domains, which may anchor this protein in the WSSV envelope (Van Hulten et al., 2002). Genome-based diagnostic methods such as in situ hybridization (Chang et al., 1996), PCR (Lo et al., 1996) and real-time PCR (Durand et al., 2003) have been developed for detection of WSSV. Serological- based diagnostic methods using polyclonal antibody (Nadala and Loh, 2000; You et al., 2002) and monoclonal antibodies specific to WSSV (Poulos et al., 2001; Anil et al., 2002; Lui et ∗ Corresponding author. Tel.: +662 664 1000x8515; fax: +662 260 0127. E-mail address: paisarn@swu.ac.th (P. Sithigorngul). al., 2002; Chaivisuthangkura et al., 2004) have been developed. However, in most cases the detection sensitivity was still lower than that of PCR. Even though the whole viral particles were used as immunogen, all monoclonal antibodies obtained were specific only to the VP28 structural protein (Poulos et al., 2001; Anil et al., 2002). Since VP19 is located in the envelope, it would be an additionally suitable target for detection of WSSV by sero- logical assays. In order to obtain antibody specific to VP19, the expression of recombinant VP19 was used to obtain sufficient amount of antigen, instead of laborious purification of WSSV particles. The antibody against VP 19 was expected to be used in combination with antibody specific to VP28 in order to improve the sensitivity of various immunoassays for WSSV detection. 2. Materials and methods 2.1. Viral and DNA preparation Natural white spot syndrome virus (WSSV) infected P. mon- odon was obtained from a farm at Nakornsrithamarat Province, 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.11.012