Development And Identification Of Monoclonal Antibodies Against Hemagglutinin Of H7N9 Avian Influenza Virus

The H7N9 subtype avian influenza(AI)has brought huge economic losses to poultry industry in China since it was found in China's Yangtze River Delta in 2013.The analysis of genetic evolution of the viral genome and pathogenicity showed that the hemagglutinin(HA)antigen of partial low-pathogenic H7N9 subtype avian influenza virus(AIV)had changed,which had developed from non-pathogenic or low-pathogenic to high-pathogenic to poultry.In addition,it has been reported that H7N9 subtype AIV can infect human,causing serious symptoms of lower respiratory tract infection and posing a serious threat to public health security.The H7N9 subtype AIV binds to receptors of susceptible cells through HA and enters the cells to replicate.The infected organism can produce hemagglutination inhibition(HI)antibody.At present,monoclonal antibodies(McAbs)are widely used in the rapid clinical diagnosis of diseases because of their high homogeneity and specificity.Therefore,the development of specific McAbs of H7N9 AIV HA has important application value.In this study,SPF chicken embryos were used to propagate the low pathogenic H7N9 subtype AIV,and the allantoic fluid was collected and inactivated by adding the final concentration of 0.1%formaldehyde solution.Then,the virus was crudely purified by ultracentrifugation.The BALB/c mice were inoculated with the initially purified inactivated virus as the immune antigen,and each mouse was immunized for 4 times with a dose of 50 ?g of virus protein each time.For the first time,the back subcutaneous multipoint injection was carried out after the mixed emulsification of Freund's complete adjuvant and virus protein.The subsequent immunization was done by the back subcutaneous multipoint injection with an equal volume of Freund incomplete adjuvant and virus protein.After 4 immunizations,the titer of indirect ELISA antibody in mice exceed 1:10000.The spleen cells from the immunized mice were fused with mouse myeloma cells(SP2/0).After fusion,the hybridoma cells were screened by indirect ELISA,and 166 positive pores were obtained.In order to obtain the monoclonal antibody hybridoma cell line against the HA of H7N9 AIV,the HI test was used to further screen the above positive pores,and the HI positive clones were subcloned by the finite dilution method.After 2 screening and subcloning,a total of 14 hybridoma cells secreting monoclonal antibodies for H7N9 AIV HA were finally obtained,which were named 2A4,2A8,2A10,4D1,4G2,5A6,5B8,5D1,5F1,5B8,4G2,4D1,4G2,4D1,4G2,5A6,5B8,5B8,4G2,4G2,5B8,4G2 and 4D1.The 7 hybridoma cell lines with HI titer exceed 4 log2(2A10?4G2?5A6?6A7?6D1?6F2 ? 6G2)were selected to prepare mouse McAb ascites,and the subclasses were further determined.The results showed that 4(2A10,6A7,6F2 and 6G2)of 7 strains were IgM,and the other 3 strains(4G2,5A6 and 6D1)were IgG1.These 3 IgG-secrecting hybridoma cell lines were serially passaged to test their stability,and the HI titer of the 20th generation's cellular supernatant had no markedly changes,meaning that these hybridoma cell lines had good stability.The McAb ascites,containing 3 strains of IgG,were purified by Protein G affinity chromatography medium,and then were further identified by HI test,indirect immunofluorescent assay(IFA),immunocytochemical assay and neutralization test.The results of HI test showed that the titers of the 3 strains of McAbs were both more than 10 log2;these 3 strains of McAbs could only react with H7N9 subtype AIV,other than H5 and H9 subtype AIV and Newcastle disease virus(NDV);4G2 and 5A6 strains showed good compatibility and could react with all the 12 strains of H7N9 subtype AIV,but 6D1 strain could only react with 10 strains of the virus.In the indirect immunofluorescence assay,all 3 strains of McAbs could react with CEF cells infected with H7N9 subtype AIV,and green fluorescence could be observed under fluorescence microscope.In immunocytochemical assay,all 3 strains of McAbs could detect AIV in CEF cells infected with H7N9 subtype AIV,and brown color is observed under microscope.In the chicken embryo neutralization test,all 3 strains of McAbs could react with the low pathogenic H7N9 subtype AIV,causing the virus to lose the ability to infect the chicken embryos;50%protective doses(PD50)of 5A6 to low pathogenic H7N9 AIV was 10-3 42,the PD50 of 4G2 was 10-2.82,and the PD50 of 6D1 was 10-2.91,showing good neutralization characteristics.This study successfully screened the specific McAbs to the H7N9 AIV HA,which laid the foundation for the later establishment of the serological detection method for H7N9 subtype AIV and the in-depth study of the structure and function of the H7N9 AIV HA antigen.