Development Of Immunogolloidal Strip Assay For Detecting The Japanese Encephalitis Virus

Japanese encephalitis(Japanese encephalitis,JE)wsa caused by Japanese encephalitis virus(JEV).It was an important mosquito-borne zoonotic infectious diseases,and could cause pregnant sows abortion,stillbom or mummified fetuses.JEV also caused boar unilateral testicular inflamed and other symptoms of reproductive of failure,especially impact on piglets.It had huge economic losses to the pig industry.It was of great significance to made a diagnosis for JEV timely and quickly. In this study, based on gold immuno-chromatography assay, a primary exploratory development was done to rapid diagnosis test strips of JEV. In this study, the main contents were as follows:1、The preparation of the Japanese Encephalitis monoclonal antibodiesIn this study,the preparation of monocolonal antibiotics by induced in mice ascites.the inject advanced sensitized by liquid paraffin BALB/C mice,inducing mice to produce ascites containing the monoclonal antibody with JEV-1and JEV-3hybridoma cell that prepared by our laboratory.The concentration of monocolonal antibody was1.953mg/mL and2.637mg/mL after purification by bitter-ammonium sulfate,and the two chains was clear by SDS-PAGE electrophoretic analysis, the monoclonal antibody which was purificed used to marker the gold particles.2、primary development and evaluation for immuni chromatograph test stripPrepared diameter25nm colloid gold with Tri-sodium citrate reduction method. In naked vision, the solution was wine red, transparent and clear, and in electron microscope, the particles were well-distributed and same size, and there’s a full and small absorption peak in522nm.results proved that the optimized pH of colloidal gold markerd was8.5and the minimum markerd was15g/mL.In the NC membrane peridium the purfied monoclone antibody as T test line,0.8mg/mL. Anti-mouse IgG as C control line,lmg/ml.The NC membrane was closed30min by PBS(contain1%BSA,2.5%sucrose,0.5%Tween-20) after peridium. The gold marking antibody was diluted by PB(contain1%BSA)peridium fiberglass as gold pad.Put the sample pad,gold pad,NC membrane,absorb pad stick on the PVC backpiane in order. Primary research showed that constructing JEV test strip with immuno-chromatography assay is feasible, it will have a favorable prospect after further improvement with package of the rapid test strips together.