Development And Preliminary Application Of The Colloidal Gold Test Strip Against Canine Parvovirus

Canine parvovirus disease(CPVD)is an acute and severe infectious disease caused by Canine parvovirus.The natural infection host of the disease is mainly dogs,especially puppies,which are more susceptible.The clinical symptoms are mainly manifested as hemorrhagic enteritis and myocarditis.The rapid spread of the disease,the extremely high morbidity and mortality rates have caused huge economic losses to the development of the dog industry and economic animal breeding industry.Therefore,the establishment of a rapid and accurate diagnostic method is of great significance for the prevention of CPVD.In this study,purified CPV and Freund's adjuvant were used to emulsify and immunize female BALB/c mice.The spleen cells of the immunized mice were fused with myeloma cells(SP2/0)by cell fusion technology.Indirect ELISA method was used to screen hybridization.The tumor cells were screened three times by subcloning,and three cell lines that specifically reacted with CPV were selected and named as 1D9,3G9 and 4F6,respectively.The results of subclass identification showed that the heavy chains of 1D9 and 3G9 were IgG1,the heavy chain of 4F6 was IgG2b,and the light chains were kappa chains.The chromosome number of hybridoma cells was determined by the colchicine method.The chromosome numbers of the three monoclonal antibodies were 92,94,and 98,respectively.1D9,3G9 and 4F6 were inoculated into the abdominal cavity of mice to prepare ascites.The titer of ascites was 10~4-10~5 by indirect ELISA.The neutralization experiment showed that the neutralization titer of4F6 ascites to completely neutralize the virus was 1:512.The crossover test showed that all three monoclonal antibodies can specifically bind to CPV,without cross-reactivity with other common canine viruses,with good specificity.Furthermore,the obtained monoclonal antibodies are used to prepare colloidal gold immunochromatographic test strips.The purified monoclonal antibody 3G9 was used as the gold labelled antibody,4F6(0.8 mg/ml)was used as the detection line,and goat anti-mouse IgG(1 mg/ml)was used as the quality control line to assemble the test strip.The test strip can detect the lowest CPV concentration of 0.0062 mg/mL,and has no cross reaction with canine distemper virus(CDV),canine coronavirus(CCV),canine adenovirus type II(CAV),etc.Finally,30 clinical samples suspected of CPV infection were tested using assembled test strips and PCR technology.The results showed that the positive coincidence rate was 92.3%(24/26)and the negative coincidence rate was 100%(4/4),so the overall coincidence rate was 93.3%[(24+4)/30].The results show that the colloidal gold immunochromatographic test strip prepared in this study can quickly and specifically detect CPV in clinical samples and can be used for on-site diagnosis of CPVD.In summary,in this study,three monoclonal antibodies with high titer and good specificity were successfully prepared,and the monoclonal antibodies were used to prepare colloidal gold test strips.The test strips were sensitive,specific,and fast.Compared with PCR technology,it has a high coincidence rate and provides technical means for the diagnosis and immune prevention of CPV in China.