Improvement of immunodetection of white spot syndrome virus using a monoclonal antibody specific for heterologously expressed icp11

Abstract

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.

Enhancement and confirmation of white spot syndrome virus detection using monoclonal antibody specific to VP26

“…The WSSV detection sensitivities of these MAbs using dot blotting are comparable to that of various MAbs specific to other structural proteins including VP28 and VP19 (Chaivisuthangkura et al 2004(Chaivisuthangkura et al , 2010 but lower than that of MAbs specific to ICP11, a nonstructural protein of WSSV (Siriwattanarat et al 2013). The WSSV detection sensitivities of these MAbs using dot blotting are comparable to that of various MAbs specific to other structural proteins including VP28 and VP19 (Chaivisuthangkura et al 2004(Chaivisuthangkura et al , 2010 but lower than that of MAbs specific to ICP11, a nonstructural protein of WSSV (Siriwattanarat et al 2013).…”

“…In a previous study, the detection limits of MAbs specific for VP28 were 500 pg lL À1 (Anil et al 2002), 400 pg lL À1 (Liu, Wang, Tian, Yin & Kwang 2002), 140 pg lL À1 or 5 fmole per spot (Chaivisuthangkura et al 2004), while the detection limits of MAbs specific for VP19 were approximately 70-280 pg lL À1 or 1.2-5 fmole per spot (Chaivisuthangkura et al 2010) and 0.7-5 fmole per spot for ICP11 (Siriwattanarat et al 2013). In a previous study, the detection limits of MAbs specific for VP28 were 500 pg lL À1 (Anil et al 2002), 400 pg lL À1 (Liu, Wang, Tian, Yin & Kwang 2002), 140 pg lL À1 or 5 fmole per spot (Chaivisuthangkura et al 2004), while the detection limits of MAbs specific for VP19 were approximately 70-280 pg lL À1 or 1.2-5 fmole per spot (Chaivisuthangkura et al 2010) and 0.7-5 fmole per spot for ICP11 (Siriwattanarat et al 2013).…”

“…Nucleic acid from the same shrimp homogenate was also extracted with a High Pure viral nucleic acid kit (Roche Molecular Biochemicals, Indianapolis, IN, USA), serially diluted with uninfected shrimp nucleic acid and tested for WSSV by onestep PCR using ICP11 11FNde (5 0 -CAT ATG GCC ACC TTC CAG ACT G-3 0 ) and ICP11 11RTYB (5 0 -GAA TTC TGT TGT TGG CAC AAT C-3 0 ) as primers to yield a PCR product of 252 bp (Siriwattanarat et al 2013).…”

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV‐2L)

“…; Siriwattanarat et al . ). In this study, we developed and characterized two mouse monoclonal antibodies (GIV‐2L‐mAb‐2 and GIV‐2L‐mAb‐18) that are specific to the GIV‐2L protein.…”

Production and characterization of a monoclonal antibody against a late gene encoded by grouper iridovirus 64L

“…; Siriwattanarat et al . ). To understand the molecular mechanism of GIV pathogenesis, the expression pattern and assay tools of viral genes must be developed and evaluated.…”