Preparation Of Monoclonal Antibodies Against HA Protein Of H1N1 Swine Influenza Virus

Swine Influenza(SI) is a highly exposed acute infectious respiratory disease of swine,caused by Swine Influenza Virus(SIV) which is orthomyxoviridae type A influenza virus. At present there has been many subtypes of SIV, consisting of H1N1 ? H1N2 ? H1N7 ?H2N3?H3N1?H3N2?H3N6?H4N6?H5N1 and H9N2. The Avian-like H1N1 subtype SIV has played an important role in the prevalence of SI of all kinds of subtypes in China.In the research, cultivating the MDCK cells for 72 hours which is inoculated by H1N1 subtype SIV and collect the supernatant after cytopathic effect. The SIV was purified by sucrose gradient centrifugation. To establish indirect ELISA method,for detection of antibodies of Avian-like H1N1 subtype SIV and screening positive hybridoma. The concentration of coated antigen is 5.725?g/ml, 4? for night. Negative and positive serums were diluted as 1:200; The best reaction condition of samples and enzyme labeled second antibody are 37?for 1h. The cross-reaction of established method with positive serums of H3N2 SIV, PRRSV, HCV and PRV is bad. In conclusion this method established by indirected ELISA is specific simple, rapid and accurate.Inactivate purified virus with 4% formaldehyde and 6-8-week BALB/C female mice are injected at multiple sites subcutaneous at 100?g per mouse after mixed with freund's adjuvant emulsion. Secondary Immunization with the same dose after two weeks. After secondary immunization collecting the blood from mice and measure the serum antibody titers with ELISA method. Strengthening immunization with mice of high antibody titers; collecting spleen cells which must be mixed with SP2/0 cells to make the hybridoma cells. Checking titers of the supernatant of hybridoma cells and screening positive cells holes. Cloning and cultivating positive cells with a limited dilution method to make 2 special hybridoma cells of H1 SIV, as 1D9,3C7. The result was showed two strains of monoclonal antibodies can react specificity with H1N1 SIV but with other subtypes of SIV or porcine pathogen. What's more,3C7 is a special sheet resistance against HA protein and which can provide material insurance for establishing the detection method of H1N1 subtype SI.