Preparation And Identification Of Monoclonal Antibodies Against The Hemagglutinin Of H5 And H9 Subtype Of Avian Influenza Virus

Avian influenza(AI) caused by A type avian influenza virus has raised great concerns for its economic importance to poultry industry and potential threat to the public health.The monoclonal antibodies against the hemagglutinin of H5 and H9 subtype AIV will be widely used in the quick diagnosis of AIV infection.Monoclonal antibodies invented in the 1970's is a high-tech of biology. Monoclonal antibodies are produced by cell fusion technology. In contrast to polyclonal antibodies, monoclonal antibodies have following virtues.identical structures,uniform component,high specificty,animate bio-activity and fewer crossing reactivity.In recent years,avian influenza has become one of the most contagious disease of poultry,so monoclonal antibodies of AIV are prospective in this study,with the purpose of preventition and diagnose.In this study,The inactivated vaccine of H5 subtype of AIV and the activated vaccine of H9 subtype virus emulsified in freund's complete adjuvant were chosen to immunize the 6-week-old female Balb/c mice with 200μg, 180μg respectively at the first time.Fourteen days later,the same antigen emulsified in in freund's incomplete adjuvant were intraperitoneally.and three days after final immunization,Splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells.Keep it in the bloom of proliferation before cell fusion.Feeder cells should be prepared before cell fusion.Kill one or more BALB/c mouse and draw its celiac macrophage and lymphocyte,then pave them in the cell culture wells.These should be operated one day before cell fusion.When cells have paved the whole cells,select posive cells using indirect ELISA.And then proliferate the positive cells. Subclone the positive cells by limiting dilution for three times to get identical cells.And the inject them into mouse abdominal cavity.Retrieve ascites in the fifteenth day.One hybridoma cell lines named A4G12 against H5 subtype of AIV and another one hybridoma cell lines named B7D10 against H9 subtype of AIV were obtained . By using indirect ELISA and HI to screen hybridoma cells, The mAbs against H9 subtype of AIV had the HI activity cells. The titers of their ascitic fluids were l:6400, 1:12800 respectively in indirect ELISA. titers.By using the immunoglobulin subtypes kit, A4G12 and B7D10 were identified to be IgG1.The McAb against H5 subtype avian influenza virus named A4G12 has no HI activity, but the specificity result of McAb named A4G12 which react to others virus by indirect ELISA method suggest the McAb had good specificity to H5 AIV so it can be a tool for the detection of AIV strains, serological investigation.The McAb named B7D10 also have good specificity to H9 AIV and what's more it has HI activity can be used to verdict the H9 subtype AIV from suspicious AI case. The McAb B7D10 can be used to establish ELISA method of detect the influenza virus of H9 subtype or help to develop the test paper with gold colloid marked to detect H9 AIV.