Preparation Of The Monoclonal Antibody Against Canine Influenza Virus And Establishment Of Its ELISA Detection

Canine influenza(canine influenza,CI)is a canine respiratory tract infection caused by A-type canine influenza virus(CIV).Influenza A virus has a wide range of hosts,such as humans,horses,swine and poultry and other animals.CIV is mainly transmitted through the air,but through the digestive tract or contact with the virus pollution of various items is also an important way of transmission.In the past it was generally believed that canine were not susceptible to influenza A virus,but over the past decade,canine were found to be infected by different subtypes of influenza virus,and canine often had clinical symptoms such as sneezing,coughing,fever and runny nose and the mortality rate was 1%to 5%.At present,H3N2 subtype canine influenza virus is widely popularin China,South Korea and other Asian countries and North America.The influenza virus has been shown to break through the barrier between species and infect different species of animals.As human companion animals,canine are in close contact with humans.Although it has not been confirmed whether the canine flu virus can infect humans and other mammals,the establishment of an efficient and convenient detection method still has important public health significance.At present,the methods of detecting canine influenza virus include virus isolation,agar diffusion(AGP)test,fluorescent antibody test,hemagglutination(HA)and hemagglutination inhibition(HI)test,nucleic acid probe technology and RT-PCR detection.Compared with these methods,ELISA is more simple,with higher sensitivity and specificity,ELISA has a lower cost and can simultaneously detect a large number of samples.The aim of this study was to prepare anti-canine influenza monoclonal antibody and establish a CIV sandwich ELISA assay to provide a valuable reference for the rapid detection and diagnosis of CIV.1.Establishment of the hybridoma secreting monoclonal antibodies against canine influenza virusBALB/c mice were immunized with the purified CIV antigen,and the prepared spleen cells were fused with well-behaved SP2/0 myeloma cells to obtain two monoclonal antibody(McAb)hybridoma cell lines(1F12 and 2A3)that capable of stably secreting anti-CIV and the antibody subclasses are IgM2bK.the two hybridoma cells were injected into the mouse abdominal cavity,the resulting antibody titer reached 105 and 106,respectively.Indirect immunofluorescence assay(IFA)showed that two monoclonal antibodies reacted specifically with CIV.Immunoblotting(IFA)showed that two monoclonal antibodies reacted specifically with other canine viruses.The two monoclonal antibodies strains can produce the specific protein bands with CIV.Hemagglutination inhibition test(HI)showed that 1F12 ascites hemagglutination titer was 210and 2A3 without hemagglutination titer.The results of the virus neutralization test showed that the neutralization titer of 1F12 was 12800 and 2A3 had no neutralizing activity.2.Establishment of the sandwich ELISA for detecting canine influenza virusTo establise the sandwich ELISA assay for detecting canine influenza virus,the monoclonal antibody 1F12 was labeled with horseradish peroxidase as the antibody and the monoclonal antibody 2A3 was purified as a coated antibody.The results showed that the specificity of the double antibody sandwich ELISA method was good,and there was no cross reaction with several other dog viruses.The repeated test was performed with different batches of ELISA strips,and the inter-assay coefficient of variation was less than or equal to 9.3%.Thirty-four clinical samples were tested by ELISA and RT-PCR.The results were consistent(23 were positive and 11 were negative).The double antibody sandwich ELISA assay established in this study has good specificity,sensitivity and repeatability,and can be used for the clinical diagnosis.