Preparation Development Of A Colloidal Gold Strip For Rapid Detection Of Infectious Pancreatic Necrosis Virus

Infectious pancreatic necrosis virus(IPNV)is a serious viral infection,it has particularly visible infectious and pathogenic to juveniles of trout and salmon,highly pathogenic strains can cause death 70 percent of juveniles within two months.It is mainly harm the juveniles of river fins,fin rainbow,brown tie,silver squatting and atlantic frogs and the mortality as high as 90%.Spreading of IPNV through eggs vertical,fish which resistance over infected can no longer resistant to the disease,but it can become lifelong carriers of the virus source of new infections,it caused the proliferation bring disaster of loss to the fish farming industry.The virus is the detection target countries for importing and exporting of fish.In this study,the anti-IPNV monoclonal antibody and anti-IPNV VP2 recombinant protein were combined with double-antibody sandwich principle to establish a colloidal gold immunochromatographic strip to detection of IPNV.The gold-labeled antibody coated on glass fiber membrane,the anti-IPNV VP2 protein polyclonal antibody coated on nitrocellulose test strip connector as a detection line,goat anti-mouse IgG antibody as a control line,in the film the other end of the absorbent was sample pad of absorbent paper.When the sample is added to the sample pad,the liquid flowed to the colloidal gold conjugate pad,and then flows to a nitrocellulose membrane and eventually flows to absorbent paper pad.If the sample contains IPNV,the test line will appear at the visible red,if the sample is free of IPNV,it does not appear at the visible red.Using bitterness-saturated ammonium sulfate and affinity chromatography purified serum antibodies using bitterness-saturated ammonium sulfate purified monoclonal antibody ascites.The purified concentration of serum antibodies was 10.014mg/ml,titer is 1.0 × 106;the concentration of ascites antibody is 2.071 mg/ml,titer is 1.0 × 106.Ascites antibodies and serum antibodies with good reactogenicity identified by Western Blot.Colloidal gold which diameter was 25nm was prepared by citric acid sodium three sodium method,the liquid was red,clear and bright and also had clear halo observed in daylight conditions;after acetate stained by TEM showed that colloidal gold particles had the same size,shape were evenly distributed,no oval and polygonal,taked multi-point measurement of the average diameter,the diameter was 25nm.Using whole wavelength UV spectrophotometer detection of colloidal gold particles,scan peak width was narrower,indicated that colloidal gold solution prepared with good dispersion.And detects the maximum absorption peak was 535nm,the maximum absorption peak absorbance value was between 1.2 and 1.3,indicated that colloidal gold particles prepared in this experiment had a good single dispersion,and was suitable to marker protein.Preparation of gold-labeled probes optimum pH value was 6.5,the best mark concentration was 36?g/ml.And using of differential centrifugation prepared colloidal gold probes.Identified various film strip models and other optimization conditions:membrane treatment of glass fiber for this study was 0.01 M pH 6.5 PBS(1%BSA,0.2%Tween-20),treatment liquid of sample pad was 0.01M pH6.5 PBS(10%sucrose,2.0%BSA,1.0%Tween-20,0.3%polyvinylpyrrolidone),treatment liquid of absorbent pad was 0.01 M pH 6.5 PBS(containing 2%BSA,1.0%Tween-20,2.5%sucrose,0.3%polyvinylpyrrolidone),suspension of gold labeled antibody was the pH 6.5 100ml of deionized water system(containing 0.2%BSA,0.5%sucrose,0.1%trehalose,0.05%casein,0.01%PEG2000,0.01%PVP-40),coating solution was0.01M pH6.5 PBS,blocking solution was 0.01M pH6.5 PBS(1%BSA),blocking time was 10min.The colloidal gold strip prepared in this study had no cross-reactivity with IHNV,the sensitivity of IPNV is 40TCID50-4.03,and the test strip were stored at 4 ? for 30d also could stability detected the IPNV.Different batches had good reproducibility,the test results of clinical samples was same with the RP-PCR.The colloidal gold immunochromatographic strip could not only fast and efficient detected IPNV,and did not required professional operators,the sample could be detected whether contain IPNV within 10 minutes.And the test strip can be widely used for its long time to save and simple operation.