Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(1) expression vector for the production of the recombinant VP28 protein.A puri-¢ed recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit.Speci¢c immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was con¢rmed by aWestern blot.The speci¢city of this polyclonal anti-rVP28 antiserum to detect the presence of the virus in WSSV-infected Penaeus monodon was veri¢ed using a immunodot blot assay.Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3^3 0 diaminobenzidine tetrahydrochloride (DAB).Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph.On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR-positive WSSV-infected shrimp samples.Immunodot blot was found to be equivalent to ¢rst-step PCR sensitivity to detect WSSV particles estimated to contain 1.0 Â 10 5 viral DNA copies.