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White spot syndrome virus (WSSV) is one of the major and most serious pathogens of shrimp disease [2, 8, 11, 13]. The virulence of WSSV is very high, resulting in mortality rates from 90 to 100% within 3 to 7 days of infection and leading to complete devastation of the shrimp culture industry. WSSV belongs to a new virus family, Nimaviridae, under a new genus Whispovirus [7], which is an enveloped virus with a 305-kb double-stranded circular DNA genome [14].
Due to the lack of an adaptive immune mechanism in crustaceans, all efforts are directed towards the development of diagnostic tests for rapid and efficient detection of WSSV at early stages of the disease in order to minimize losses. Several techniques such as histological observation, in situ hybridization, PCR, western blot analysis or loop-mediated isothermal amplification (LAMP) have been developed [6, 9]. Some of them are laborious and time-consuming, while others have practical limitations to their wide spread application because of high cost and the need for special equipment and well-trained skilled personnel. Therefore, an antibody-based assay presents an attractive alternative, providing a simple and low-cost detection system with high specificity and optimal sensitivity for disease monitoring. Immunological diagnosis based on anti-WSSV immune serum as well as monoclonal antibodies (MAbs) have been used successfully to detect WSSV antigens [10, 12], but most of these MAbs were produced with purified WSSV or native envelop proteins of WSSV.
Since the purification of WSSV from infected shrimp is a laborious and time consuming task, we considered whether MAbs could be produced with recombinant envelope proteins from WSSV. A MAb specific to recombinant VP19 was developed to detect WSSV [1], but no MAb specific to recombinant VP28 was reported before. VP28 is one of the most abundant envelope proteins of WSSV [16] and is crucial for virus entry and vaccine-induced protection [5, 17]. Hence the present article reports the development of MAb against recombinant VP28 (rVP28) that can detect WSSV rapidly and sensitively at the early stages of WSSV infection.
Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus *
- Received Date: 17 May 2011
- Accepted Date: 29 June 2011
Abstract: The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21. After induction, the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production. It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection. Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay. Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.