Development Of Kits For Detecting White Spot Syndrome Virus Of Shrimp And Their Application

The disease of white spot syndrome virus (WSSV) is one of the main factors that obstruct the development of shrimp aquaculture, there is no highly effective drug or treatment for this virus disease until now, therefore, the research for a rapid, simple, convenient and specific detection kit to make informed decisions in the field is very imperative in order to implement preventive or control measures. Development of kits for detecting WSSV of shrimp and their application described in this paper only meets this urgent need.A great many monocolonal antibodies, 12 of them coming from this experiment and the other parts from our laboratory which were produced previously, were selected by means of Immunofluorescence assays. 8 strains of hybridomas producing stronger positive monocolonal antibodies against WSSV of shrimp were gained and ascitic fluid was made in order to get high specificity and affinity of anti-WSSV monocolonal antibodies. Then those anti-WSSV monocolonal antibodies were purified and used in the development of kits for detecting WSSV of shrimp.In this paper, we established an ideal Dot-immunogold filtration assay protocol that is more rapid, simple, accurate and not false positive result for the specific detection of WSSV. Nitrocellulose membrane as a support of samples of WSSV and anti-WSSV monoclonal antibodies (2E6 and 2A3) labeled red color colloidal gold developed Dot-immunogold filtration assay for detection of WSSV. Affected by filtration and condensation, the antigen and antibody reaction was enabled to go on rapidly, so the whole process can be completed in 3 min and without incubation or any equipment. A reddish dot indicating positive result was obvious to the naked eye. Comparing with Dot-blot nitrocellulose enzyme immunoassay, the detection limit of Dot-immunogold filtration assay was similar to it, but the Dot-immunogold filtration assay was more rapid, convenient, accurate and not false positive result. It can be carried out in the shrimp farms only with a colloid gold kit.A rapid, single step, sensitive and specific Immunoaffinity chromatograpbic stripe was developed for detecting WSSV by using anti-WSSV monoclonal antibodies (2E6 and 2A3) labeled with red color colloidal gold and anti-WSSV monoclonal antibody (4G9) as capture antibody on nitrocellulose membrane. The principle of "sandwich" using the distinct monoclonal antibodies for the rapid detection of the antigen was adopted, so the test process can be completed within 5 min without incubation or any equipment. A reddish colour at the site of immune reaction on nitrocellulose membrane indicating positive result was obvious to naked eyes. Comparing with Dot-blot nitrocellulose enzyme immunoassay, the sensitivity of Immunoaffinity chromatographic stripe was similar to it, but Immunoaffinity chromatographic snipe was more rapid, simple, convenient and without false positive results. It can be done pond side just using a test stripe kit.The use of a visible colloidal gold particles conjugating with antibody instead of an enzyme conjugating with antibody in Dot-blot nitrocellulose enzyme immunoassay ensures that Dot-immunogold filtration assay and Immunoaffinity chromatographic stripe avoids the disturbance of endogenous enzyme in tissus, so there are not false positive results in Dot-immunogold filtration assay and Immunoaffinity chromatographic stripe. The protocol of filtration and chromatographer used in the detection of WSSV makes that the reaction in the two kits is rapider than Dot-blot nitrocellulose enzyme immunoassay and can be completed within 5 min.Using Dot-immunogold filtration assay and Immunoaffinity chromatographic stripe to discover the disease of WSSV, then how can we control and prevent the epidemic of WSSV? The relationship between infection of WSSV and envelope integrity was just studied in order to accumulate more experimental data for cutting down the epidemic of WSSV and discovering a new way to control the disease of WSSV. The results showed that the virus with broken envelope made by low osmotic pressure could infect crayfish (Cambrus proclarkit) in vivo and vitro. Additionally, anti-WSSV monoclonal antibody (4G9) recognized epitopes on envelope of virus and the other one (2A3) recognized epitopes on the capsid were used to neutralize the infection of WSSV respectively, the results showed that the anti-WSSV monoclonal4antibody (4G9)had neutralizing effect and the other one (2A3) hadn't in vivo. Finally, the pure capsides without any envelope around them was used to repeat the infecting test above, and the result showed that the pure capsides couldn't induce infection in vivo, which suggested that WSSV's infection only depended on the quantity and activity of envelope's function proteins connecting with infection. As long as those function proteins possessed activity and had adequate quantity to combine with the member of target cell, WSS V would induce infection, even if the envelope of WSSV was not intact, which might be one of the main reasons why it's so difficult to control the epidemic of WSSV.