Preparation And Preliminary Application Of Immune Colloidal Gold Strip For Diagnosis Of Goose Paramyxovirus Disease

Goose paramyxovirus disease also known as goose newcastle disease, is a viraldisease caused by the goose source paramyxovirus designated avian paramyxovirus1with high morbidity, mortality and infection rates. ND outbreaks in geese with severeclinical signs was first reported in1997in Jiangsu province of China then was foundepidemic in many regions of the country within several years causing great economiclosses which is seriously threatenning the healthy development of geese industry.Diagnosing goose paramyxovirus disease promptly to cull infected geese in theshortest time is significant for prevention more infection and greater economic losses.Commonly methods used for goose paramyxovirus disease diagnosis includingisolation and identification of the virus, HA-HI test, agar diffusion test, ELISA,immunofluorescence, RT-PCR, real-time quantitative PCR are inconvenient to use onfield. Colloidal gold immunochromatography test strip using nitrocellulose membraneas the reaction carrier, colloidal gold as the reaction tracer and immunoadsorption asworking principle has advantages of simple to use, visible to the naked eyes, hence isconsidered suitable for rapid diagnosis of goose paramyxovirus disease on field.In this study, the NA-1strain of goose source paramyxovirus which werepurified by sucrose linear density gradient centrifugation after embryo proliferationwere used for immunization of6-8weeks’ BALB/c mice in assordance withconventional procedures. Mice with the serum titer of1:120800were chose to provideimmune spleen cells for cell fusion with SP2/0myeloma cells in the logarithmicgrowth phase. An indirect ELISA method was established for screening positivehybridoma cell lines. Three lines of hybridoma cells named3E9、2B8、1D8with thetiter of1：6400、1：3200、1:3200respectively were obtained after limited dilutioncloning more than three times.The average chromosome numbers of3E9、2B8、1D8cells was respectively98、91and87in line of the hybridoma cells. Specificity of the three monoclonal antibodies were identified by indirect ELISA with9strains of virus, including goosesource paramyxovirus NA-1、national standard virulent strain of chicken newcastledisease F48E9and chicken source newcastle disease viru strain La Sota, and otheravian disease virus, such as AIV、IBV、IBDV and MDV. All of the three monoclonalantibodies has positive reaction to strains of goose paramyxovirus NA-1、nationalstandard virulent strain of chicken newcastle disease F48E9and chicken sourcenewcastle disease viru strain La Sota, while was negative to other virus. Antibodysubclasses of3E9,2B8and1D8was IgG1， IgM and IgM respectively. Ascitesprepared by3E9hybridoma cells had a ELISA titer of1:160000. Hemagglutinationinhibition test of3E9ascites to the7strains of virus mentioned before was taken. TheHI titer of3E9to NA-1、F48E9、La Sota strains respectively was2¹⁴、2¹²、2¹¹，andno hemagglutination inhibition was found to other4strains of virus. Result of theindirect immunofluorescence assay has shown that the3E9ascites was able torecognize the NA-1、F48E9、La Sota strains specifically. The concentration of theascites after purified was2.13mg/mL, and the ELISA titer was1:128000. Polyclonalantibody anti-NA-1with the concentration of4.26mg/mL and the titer of1:640000was prepared by two adult rabbits.Immune colloidal gold strip for diagnosis of goose paramyxovirus disease wasprepared by using3E9McAb labele colloidal gold, rabbit anti-NA-1polyclonalantibody as the test line and goat anti–mouse IgG as the control line. The sensitivityof the strip was8times of the HA-HI assay for goose paramyxovirus, and2times ofthe HA-HI assay for F48E9and La Sota strains. The results of the detection to throatswabs and anal swabs from goose by this immune colloidal gold strip were88%and98%matched with the HA-HI assay of goose anti-NA-1positive serum. The immunecolloidal gold strip for diagnosis of goose paramyxovirus disease prepared in thisstudy provides a simple method for rapid diagnosis of goose paramyxovirus diseaseon field.