Preparation Of Monoclonal Antibodies Against Canine Parainfluenza Virus And Establishment Of Colloidal Gold Detection Technology

Canine Parainfluenza Virus（CPIV）is one of the main pathogens of infectious respiratory diseases in dogs.Respiratory symptoms such as fever,runny nose,and cough are common after CPIV infection in dogs,which are harmful to the health of dogs.The symptoms of this disease are similar to those of dogs infected with Canine Distemper Virus（CDV）,and it is difficult to directly differentiate the disease.Therefore,it is essential to establish a rapid,simple and accurate detection method.It was found that the sensitivity of CPIV test strips on the market was low,and there was no specific treatment for the disease at present.Therefore,this study screened neutralizing antibodies and detection antibodies at the same time,in order to provide reference for clinical treatment,monitoring and epidemiological investigation of CPIV,and to provide help for the prevention and control of canine respiratory disease.The main findings of this study are as follows:1.BALB/c mice were immunized with PEG6000 concentrated CPIV virus solution as immunogen.Cell fusion was performed as soon as the neutralization titer reached 1:10⁴after three immunizations.Using CPIV as coating antigen,the fusion cells were screened by indirect ELISA.Three positive hybridomas,named 1B12,4E3and 5F3,were successfully selected through three subcloning.The monoclonal antibodies secreted by the above three hybridoma cells were identified.All three hybridoma cells could secrete specific antibodies against CPIV.The secreted monoclonal antibodies were Ig G1 for heavy chain 1B12 and 5F3,Ig G2a for heavy chain 4E3,and Kappa for light chain of hybridoma cells.By Western-Blot analysis,4E3 was found to be an antibody to the CPIV F protein,and 1B12 and 5F3 were found to be antibodies to the HN protein.The 4E3 antibody was found to be neutralizing by neutralization assays in virus-infected cells.2.The titer of ascites produced by hybridoma cell line 1B12 was 1×10⁵,4E3 was1×10⁶,and 5F3 was 1×10⁴.Indirect immunofluorescence assay showed that the ascites of the 3 strains could only react with CPIV,and had no obvious cross-reaction with CAV-2,CDV and CPV.Two monoclonal antibodies（4E3 and 1B12）with high ascites titer were purified by octanoic acid-ammonium sulfate method and Protein G column.The heavy chain and light chain bands of the two monoclonal antibodies were clear,the purity was more than 95%,and the concentrations were 2.3 mg/m L and 1.3 mg/m L respectively.3.Using 4E3 as the labeled antibody and 1B12 as the detection antibody,the optimal reaction conditions were determined,and the double-antibody sandwich colloidal gold test strip method for antigen detection was established.The minimum detection limit of the test strip detection system established in this study was 10^1.67TCID₅₀/m L,and the sensitivity was good.It reacted only with CPIV,and there was no obvious cross-reaction with CAV-2,CDV,CPV,etc.When the test strip was placed at 37℃for 60 days,the intensity of C line and T line was consistent and the stability was good.The test strip prepared in this study and the commercial CPIV antigen detection test strip were compared with similar products.The CPIV virus solution of10^4.67TCID₅₀/m L was detected.The test strip showed clear color,and the detection line could be seen when diluted to 1:1280,and the minimum detection limit was10^1.67TCID₅₀/m L.The commercial test strip showed light color,and the detection line was visible when diluted to 1:100,but no obvious detection line was found when diluted to 1000 times,indicating that the sensitivity of the test strip established in this study was about 10 times higher than that of the commercial test strip.46 clinical samples were detected by this test strip and RT-PCR simultaneously,and the coincidence rate was 97.8%.