Development And Identification Of Monoclonal Antibodies Specific To X-19 Of H9N2 Subtype Of Avian Influenza Virus

Avian Influenza(AI)is an infection disease caused by type A avian influenza virus(AIV).It was divided into highly pathogenic and low pathogenic according to their pathogenicity.H9N2 subtype avian influenza virus is a representative virus of low pathogenic avian influenza virus.Although the lethal rate of low pathogenic avian influenza virus was not high,it will result in immunosuppression and secondary infection,reduced the laying rate and caused huge economic losses to poultry industry.The existing H9N2 subtype avian influenza diagnostic methods were traditional separation etiology,serology and RT-PCR diagnosis and etc.Those methods were disadvantaged in rapid diagnostic technique,because of the complicated process and expensive equipment requirements.Thus,the preparation of monoclonal antibodies which against X-19 of H9N2 subtype of avian influenza virus was needed.It can be used to establish simple,rapid diagnostic method for avian influenza.This experiment used X-19 of the H9N2 subtype of AIV as antigen to get the monoclonal antibodies(Mabs).X-19 of H9N2 subtype of AIV were inoculated into 10-day-old SPF chicken embryos via the allantoic cavity.After three generations of continuous inoculation,we harvested the allantoic fluid of X-19 virus.Its hemagglutination titer was 10log2.By using differential centrifugation to concentrate virus,we increased the X-19 virus HA titer of X-19 virus from 10log2 to 13log2.Using sucrose density gradient centrifugation to purify the virus,when the centrifugal stop,there was one obvious virus band between the sucrose density 30% and the sucrose density 60%.We inactivated the concentrated X-19 virus,and mixed it with Freund's adjuvant.According to the immune procedure,we vaccinated the 6 weeks of Balb/c mice.We used purified X-19 virus as the package antigen to establish the indirect ELISA method.so that,we could detected the serum antibodies titer in mice.When the titer was more than1: 102 400,we harvested the mouse spleen cells for cell fusion with NS0 cells.We used the established ELISA method to select the hybridomas,and used the limiting dilution to purifiy the hybridomas.Finally,we get six hybridoma cell lines named 1A7,2Bl1,2C9,8F3,4F3 and 3B9.Using ELISA and hemagglutination inhibition(HI)test respectively detected the supernatant and ascites of 6 hybridoma cell lines.The result showed that the HI titer of supernatant were all higher than 6log2,and the Hi titer of ascites were higher than 8 log2,except 2c9.The ELISA titer of supernatant was between1: 800 ~ 1:6400,and the ELISA titer of ascites titer were between 1:3200 ~1: 51200.Specific identification results: six Mabs only reacted with X-19 of H9N2 subtype of Avian influenza,and did not react with IBDV?H3N2?NDV?FADV.This showed that,they all were highly specificity.Test the Mabs with different H9 subtype of AIV,we found that 8F3 reacted with 4 strains of diferent H9 subtype of AIV,which could be initially determined 8F3 had a wide response spectrum,we speculated that 8F3 specific identificated a conservative sites of H9 subtype of AIV,which remained to be further test.Indirect fluorescent experiment: six Mabs had bright immunofluorescence,this showed that they had good immune activity,and had the potential to prepare a rapid detection kit to H9 subtype AIV.