The icp11 gene encoding the highly abundant DNA mimic protein of white spot syndrome virus (WSSV) was cloned into the pTYB1 and pGEX-6P-1 expression vectors and introduced into E. coli by transformation. After induction, C-terminally intein-tagged ICP11 (ICP11-intein) and N-terminally glutathione-S-transferase (GST)-tagged ICP11 (GST-ICP11) proteins with molecular masses of 64 and 35 kDa were obtained. These proteins were purified by SDS-PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific for ICP11 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei by dot blotting, western blotting or immunohistochemistry without cross-reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was approximately 0.7 fmole/spot of GST-ICP11 as determined by dot blotting. These MAbs showed stronger immunoreactivity than other MAbs from previous studies that are specific for VP28 and VP19. A combination of MAbs specific for ICP11, VP28 and VP19 increased the detection sensitivity of WSSV during early infection to a sensitivity 250 times lower than that of one-step PCR. Therefore, the MAbs specific for ICP11 could be used to confirm and enhance the detection sensitivity for WSSV infection in shrimp using various types of antibody-based assays.

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The simple immunoprecipitation method was used to isolate tilapia immunoglobulin (Ig) for immunization in order to produce monoclonal antibodies (MAbs) specific to tilapia Ig. First, the tilapia antiserum against bovine serum albumin (BSA) was prepared by peritoneal injection of BSA into tilapia, and the tilapia anti-BSA antiserum was used to precipitate BSA to form the Ig/BSA immune complex. The Ig/ BSA immune complex was then injected into Swiss mice for hybridoma production. After fusion, three hybridoma clones producing MAbs specific to the tilapia antibody were selected by dot blot and Western blot. All MAbs (101A, 59G, and 11A) were bound specifically to the heavy chain of immunoglobulin M (IgM). The MAbs 101A and 59G demonstrated twofold higher affinity than MAb 11A and the commercialized antibody. However, MAbs 11A could also bind to the heavy chain of IgM in Asian seabass, Lates calcarifer, as well. These MAbs can be used to monitor the immune responses of individual fish by indirect ELISA upon exposure to various antigens.

The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (Mr NV) 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-Mr NV; 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 Mr NV 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-Mr NV, 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 Mr NV. KEY WORDS: Capsid protein · Extra small virus · XSV · Immunohistochemistry · Macrobrachium rosenbergii nodavirus · Mr NV · Monoclonal antibody · Western blot Resale or republication not permitted without written consent of the publisherDis Aquat Org 98: [121][122][123][124][125][126][127][128][129][130][131] 2012 Genome-based detection methods with high specificity and high sensitivity for detection of MrNV include dot blot hybridization, in situ hybridization (Sri Widada et al. 2003) and 1-step RT-PCR in the form of single tests for MrNV or XSV (Sri Widada et al. 2003, Sahul Hameed et al. 2004 or multiplex tests for both viruses (Yoganandhan et al. 2005, Tripathy et al. 2006. A 2-step real-time RT-PCR method (Zhang et al. 2006) and a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method (Pillai et al. 2006, Puthawibool et al. 2010) have also been described. Although a thermal cycler is not required for RT-LAMP, the technique is still expensive and fairly complicated, and a laboratory is required. Therefore, these molecularbased techniques are not useful for pond-side detection by shrimp farmers, who require simpler and quicker disease monitoring and disease-outbreak confirmation methods that are easy to perform with high specificity and optimal sensitivity. These objectives may be achieved using immunoassay methods. Use of a polyclonal antibody (PAb) (Romestand & Bonami 2003) and monoclonal antibodies (MAbs) (Qian et al. 2006) to detect purified MrNV using sandwich ELISA and Western blotting (Sahul Hameed et al. 2011) have been described. Alth...

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