Improved immunodetection of Penaeus monodon densovirus with monoclonal antibodies raised against recombinant capsid protein
2011
Srisuk, Chutima | Chaivisuthangkura, Parin | Sukhumsirichart, Wasana | Sridulyakul, Pattarin | Longyant, Siwaporn | Rukpratanporn, Sombat | Sithigorngul, Paisarn
Penaeus monodon densovirus (PmDNV), also called hepatopancreatic parvovirus (HPV), is a pathogen that causes stunt disease in the tiger shrimp, Penaeus monodon. In this study, the gene sequence encoding the capsid protein of PmDNV was cloned into two parts: PmDNV-N (837bp) and PmDNV-C (806bp). Both PCR products were cloned into the pGEX-6P-1 expression vector and transformed into the Escherichia coli BL21 strain. After induction, glutathione-S-transferase (GST)-tagged PmDNV-N and GST-tagged PmDNV-C proteins were obtained with molecular masses of 57.2 and 57.6kDa, respectively. The recombinant proteins were purified by SDS-PAGE and used to immunize mice for monoclonal antibody (MAb) production. Three hybridoma clones producing MAbs specific to PmDNV-N and two hybridoma clones producing MAbs specific to PmDNV-C were selected by dot blot, Western blot and immunohistochemistry. All five MAbs detect PmDNV infection in shrimp by dot blot and Western blot, but only PmDNV-specific MAbs can be used to detect PmDNV infection in the hepatopancreas by immunohistochemistry. The detection limit of PmDNV-N-specific MAbs obtained in this study is similar to the most sensitive MAb characterized in a previous study, approximately 50fmolμl⁻¹ of antigen as determined by dot blot; the sensitivity of PmDNV-C-specific MAbs is twofold less than the MAbs specific to PmDNV-N. Using three MAbs specific for PmDNV-N and PmDNV-C together is twofold more sensitive than using either MAb alone. Although the detection limit of combining MAbs was 25,000 times lower than one-step PCR-based methods, using these MAbs is still useful for confirmation of PmDNV infection which is more convenient than normal histopathology confirmation.
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