A simple and rapid immunochromatographic test strip for detection of white spot syndrome virus (WSSV) of shrimp

Sithigorngul, Weerawan; Rukpratanporn, Sombat; Pecharaburanin, Nilawan; Longyant, Siwaporn; Chaivisuthangkura, Parin; Sithigorngul, Paisarn

doi:10.3354/dao072101

Cited by 71 publications

(45 citation statements)

References 12 publications

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“…3). As for other viruses (W. Sithigorngul et al 2006, P. Sithigorngul et al 2011, MAbs targeted to different epitopes on the XSV CP in combination with the MAb generated recently (Wangman et al 2012) to the MrNV CP should allow immunochromatographic strip tests to be developed for the rapid pond-side diagnosis of WTD. …”

mentioning

confidence: 99%

Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus

Longyant¹,

Senapin²,

Sanont³

et al. 2012

Dis. Aquat. Org.

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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...

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mentioning

confidence: 99%

Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus

Longyant¹,

Senapin²,

Sanont³

et al. 2012

Dis. Aquat. Org.

Self Cite

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“…For TSV, the detection limit per spot for MAbs against VP3 was 400 to 800 pg (Longyant et al 2008) and against VP2 was 400 to 800 pg of VP2 (Chaivisuthangkura et al 2010b), while that for the capsid protein of PstDNV was 300 pg (Sithigorngul et al 2009). Because the MAbs targeted different epitopes on the MrNV capsid protein, they are expected to be useful for the development of an immunochromatographic strip test for simple and rapid detection of MrNV infection, as has been reported previously for WSSV and YHV (Sithigorngul et al 2006(Sithigorngul et al , 2007(Sithigorngul et al , 2011.…”

Section: Discussionmentioning

confidence: 97%

“…These MAbs were selected and characterized using various immunological methods including dot blotting, Western blotting and im muno histochemistry. The objective was to obtain MAbs that targeted a variety of MrNV epitopes for use in the development of a simple and rapid strip test that required at least 2 MAbs, one as a captured antibody and another as a labeled antibody, that recognized different epitopes of the antigen in sandwich format (Sithigorngul et al 2006(Sithigorngul et al , 2007(Sithigorngul et al , 2011. …”

mentioning

confidence: 99%

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein

Wangman¹,

Senapin²,

Chaivisuthangkura³

et al. 2012

Dis. Aquat. Org.

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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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“…Escherichia coli BL21 with VP3-pGEX-6P-1 plasmid (Chaivisuthangkura et al 2006) was cultured in Luria-Bertani (LB) broth to the exponential growth phase and expression of the recombinant protein rVP3 was induced with 1 mM isopropyl-β-D-thiogalacto-pyranoside (IPTG) for 4 h. After centrifugation at 4000 × g for 20 min, the bacterial pellet was resuspended in 100 mM NaH 2 PO 4 , 10 mM Tris-HCl, 8 M urea pH 8, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and sonicated until a clear lysate was obtained. The lysate was resolved using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).…”

Section: Methodsmentioning

confidence: 99%

“…However, this MAb failed to detect several isolates of TSV from various sources (Erickson et al 2002, Robles-Sikisaka et al 2002. Polyclonal antibodies against recombinant proteins of VP1 and VP3 have also been developed (Chaivisuthangkura et al 2006). Results similar to MAb 1A1 immunoreactivity (Erickson et al 2005) have been observed in which anti-rVP1 antiserum failed to react in many chronically TSV-infected P. vannamei that gave positive results with anti-rVP3 antiserum by immunohistochemistry (P. Chaivisuthangkura unpubl.…”

Section: Abstract: Penaeus Vannamei · Monoclonal Antibody · Mab · Tamentioning

confidence: 99%

Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV)

Longyant

Poyoi²,

Chaivisuthangkura³

et al. 2008

Dis. Aquat. Org.

Self Cite

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A simple and rapid immunochromatographic test strip for detection of white spot syndrome virus (WSSV) of shrimp

Cited by 71 publications

References 12 publications

Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus

Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein

Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV)

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