Development Of Colloidal Gold Immunochromatographic Test Strips For The Detection Of Antibody And Pathogeny Of Pseudorabies

In recent years,swine pseudorabies has become widespread in many domestic pig farms again,which is extremely harmful to the pig industry in China.Therefore,it is very important to develop a simple,rapid,sensitive and specific method for the rapid detection of the disease and antibody.In this study,simple,rapid,sensitive,and specific colloidal gold immunochromatographic test strips were developed for the detection of antibodies and pathogeny of pseudorabies.The specific research content is as follows:1.Immunochromatographic test strips were developed for the detection of antibodies against pseudorabies.Indirect method was used in this detection system.Pseudorabies virus?PRV?was purified using sucrose density gradient centrifugation and results in one band at each of the 30%,40%,and 50%sucrose gradients.The purified virus particles were detected by PCR and ELISA which has good specificity.The concentration of the virus particles in 40%sucrose gradient was found to be the highest when determined by BCA.Therefore,the virus in this layer was used to combine with biotin-N-hydroxysuccinimide ester?BNHS?to form BNHS-PRV complex,and then the complex was labelled with the well-prepared colloidal gold particles.The Staphylococcal protein A?SPA?and the streptavidin?SA?were coated on the test line?T line?and the control line?C line?of Nitrocellulose membrane respectively.The reaction system was optimized through a series of conditions,including the treatment of sample pad,gold spray volume,BNHS-PRV complex labeling volume,SPA coating concentration and serum dilution ratio,etc.The test strip was assembled under the comprehensive optimization conditions.The results showed that the test strip had no cross reaction with PCV-2,CSFV,PRRSV and FMDV positive sera.The specificity of this method was good.The 175 clinical swine sera were tested by this test strip and commercial gB-ELISA kit respectively.The results showed that the positive coincidence rate,the negative coincidence rate,and the total coincidence rate was93.9%,83.3%and 91.4%respectively.2.Immunochromatographic test strips were developed for the detection of pathogens of pseudorabies.Double antibody sandwich method was used in this detection system.Firstly,PRV gB monoclonal antibody was labeled on the well-prepared colloidal gold particles,and then sprayed onto the binding pad.Secondly,PRV gB monoclonal antibody and the goat anti-mouse IgG was coated on the test line?T line?and the control line?C line?of Nitrocellulose membrane respectively.The reaction system was optimized through a series of conditions,including pH,the treatment of sample pad,PRV gB monoclonal antibody concentration,gold sprayed volume,and PRV gB monoclonal antibody labeling volume.The test strip was assembled under the comprehensive optimization conditions.The results showed that the test strip had no cross reaction with PEDV,PCV-2,CSFV,PRRSV and TGEV viruses and its sensitivity was 4.375×10^4.5 TCID₅₀.Twenty-one clinical samples of suspected pseudorabies were tested by this test strip and gE PCR respectively.The results showed that the positive coincidence rate,the negative coincidence rate,and the total coincidence rate was 84.6%,87.5%and 85.7%respectively.In this study,a colloidal gold immunochromatographic test strip was preliminarily developed for the rapid detection of antibodies against pseudorabies in sera.Based on the double-antibody sandwich method,a colloidal gold immunochromatographic test strip was developed for the rapid detection of pseudorabies virus.