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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A strip test was developed for detection of infectious myonecrosis virus (IMNV) using a pair of monoclonal antibodies (MAbs), called IMN7 and IMC6, that are specific for the N and C fragments, respectively, of the IMNV capsid protein. The test strips were placed in plastic cassettes and stored desiccated in sealed plastic bags. In detection assays using the test-strip cassettes, 100-μl samples of application buffer containing homogenates from muscles or pleopods of normal or IMNV-infected shrimp were applied to the cassette sample chamber. Subsequent flow through the glass-fiber pad and the nitrocellulose membrane strip led to the development of visible antibody-protein complexes within 15 min. In samples containing IMNV, viral capsid protein bound to gold-labeled IMN7 in the glass-fiber pad and the complex was subsequently captured by MAb IMC6 at the T line to form a reddish-purple band. Any unbound gold-labeled IMN7 migrated past the T line to be captured by the GAM antibody to form a band at the C line. Samples without IMNV or containing it below the test detection limit gave reddish-purple bands only at the C line. The sensitivity of the test was comparable to that of dot blot tests using single MAbs but was ~300-fold less sensitive than a one-step RT-PCR test for IMNV. Despite this lower sensitivity, the strip test has advantages of low cost, speed and simplicity (i.e., no sophisticated equipment or specialized skills required), and it is appropriate for use by farmers for pathogen confirmation when IMNV is suspected in diseased shrimp.

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VP AHPND ), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of How to cite this article: Wangman P, Chaivisuthangkura P, Taengchaiyaphum S, Pengsuk C, Sithigorngul P, Longyant S. Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease. J Fish Dis.

The capsid protein (CP) gene of extra small virus (XSV) expressed in Escherichia colias a 42 kDa glutathione S-transferase (GST)-fusion protein (GST-XCP) or a 20 kDa His 6 -fusion protein (His 6 -XCP) were purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), combined, and used to immunize Swiss mice to produce monoclonal antibodies (MAbs). Using dot blot, Western blot, and immunohistochemistry (IHC) methods, 4 MAbs specific to the XSV CP detected XSV in the freshwater prawn Macrobrachium rosenbergii without crossreaction to host proteins or to proteins of Macrobrachium rosenbergii nodavirus (MrNV) or 5 of the most pathogenic viruses of penaeid shrimp. In dot blots, the combined MAbs could detect down tõ 10 to 20 fmol µl −1 of purified GST-XCP protein, which was somewhat more sensitive compared to any single MAb. Used in conjunction with an MrNV-specific MAb, white tail disease (WTD) was diagnosed more effectively. However, the sensitivity at which the combined 4 MAbs detected XSV CP was 1000-fold lower than XSV RNA detected by RT-PCR. IHC analysis of M. rosenbergii tissue sections using the MAbs showed XSV infection to co-localize at variable loads with MrNV infection in heart and muscle cells as well as cells of connective tissues in the hepatopancreas. Since XSV histopathology remained prominent in tissues of some prawns in which MAb reactivity for MrNV was low compared to MAb reactivity for XSV, XSV might play some role in WTD severity. KEY WORDS: Immunohistochemistry · Macrobrachium rosenbergii nodavirus · MrNV · Monoclonal antibody · Capsid protein · Western blot · Extra small virus · XSV Resale or republication not permitted without written consent of the publisherDis Aquat Org 99: 197-205, 2012 198 losses of PL in hatcheries and nursery ponds within 5 d of gross signs first appearing (Arcier et al. 1999). In muscle tissue affected by WTD, MrNV is always found in conjunction with XSV . MrNV is a small (27 nm diam.) non-enveloped icosahedral virus with a genome comprising 2 singlestranded RNAs 2.9 kb and 1.3 kb in length and a capsid comprised of a single 43 kDa protein (Bonami et al. 2005). The XSV genome is a singlestranded RNA 796 nucleo tides in length that encodes a 16−17 kDa capsid protein (CP). XSV is considered to be a satellite virus dependent on the MrNV RNA-dependent RNA polymerase for genome replication , Bonami et al. 2005.Dot blot and in situ hybridization (Sri Widada et al. 2003, Hsieh et al. 2006, Wang et al. 2008, RT-PCR (Sri Widada et al. 2003, Sahul Hameed et al. 2004, multiplexed RT-PCR (Yoganandhan et al. 2005, Tripathy et al. 2006, Owens et al. 2009, real-time RT-PCR (Zhang et al. 2006), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (Pillai et al. 2006, Haridas et al. 2010, Puthawibool et al. 2010) have been developed to detect MrNV and/or XSV RNA. While highly specific and sensitive, all of these molecular detection methods are quite complicated and expensive and thus not practical for pond-side detection of the...