Establishment Of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against H9 Subtype Avian Influenza And Application In The Rapid Diagnosis Strip

AIV H9N2 was grown in chicken embryonated eggs and purified by differential centrifugation and sucrose density centrifugation.Eight-week Balb/c mice were immunized according to Kazuhiro's produre.Two hybridoma named 1H6, 3A3were generated by fusion of NSo myelomas and Splenocytes from immunized mice.The monoclonal antibody(McAb) produced was screened by ELISA and HI and proved to be IgG1, IgG2b by AGP,mouse ascetic fluid(HI 2¹⁴, 2¹⁶) was purified(protein concentration : 19mg/mL, 14.9mg/mL) and proved to react with H9N2 AIV only in HI.The result of cross-reaction in HI demonstrated that two McAbs were subtype-sepecific.The purified rabbit anti-AIVIgG was labeled with colloidal gold particals of 10nm in diameter.Then it was immobilized to nitrocellulose membrane .The two kinds of antibody concentration was optimized by the sqaure matrix test .A McAb-mediated rapid diagnosis strip was developed for detection of H9N2AIV.When we want get the result ,put it into the sample ,wait for 5 minutes.The sample with obvious red line on the strip was judged as positive for H9N2AIV,others,negative.The detection limit of purified H9N2AIV allantioic fluid of H9N2AIV were 16.56ng and 2HAU respectively.The test strip was placed in 4°Cdesiccator,after 8 month its effect of detection and releasing still hold on the line.The test strip show is easy to be justified .It demonstrated that rapid diagnosis strip was a rapid, sensitive, simple and non-lable-intensive test for detection of H9N2 subtype AIV.These key techniques and the data will help us research and develop a series of GICA strip to detect the domestic animals and poultry.