Preparation And Preliminary Application Of Monoclonal Antibodies Against HA Protein Of H7N9 Avian Influenza Virus

In 2013,the novel H7N9 avian influenza virus?AIV?infection was firstly reported in China and caused a large number of deaths.The drugs to control H7N9 virus are limited,and a rapid diagnosis technology for detection H7N9 AIV and an anti-H7N9drugs are urgent.Therefore,the following three parts of research were conducted in this study:Part I.Generation of monoclonal antibodies?mAbs?against H7N9 virus hemagglutinin?HA?.BALB/c mice were immunized with the H7N9 virus vaccine?A/ZJU01/PR8/2013?,positive hybridomas were screened by enzyme-linked immunosorbent assay?ELISA?and six mAbs were finally selected.By isotyping,4mAbs?1H10,2D5,2A9 and 2C6?belonged to the Ig G1,and 2 mAbs?2D1 and 2F5?belonged to Ig G2a.All of six mAbs showed strong affinity to HA protein.Additionally,six mAbs were specific to H7 AIVs and showed no cross-reactivity with other subtypes of AIVs by immunofluorescence assay.Part II.Development of a colloidal gold test strip and an ELISA kit to detect H7N9virus.Based on antibody characteristics and optimized pairing experiments,mAbs 2D5and 2F5 were selected as capture and detection antibody,respectively.A colloidal gold test strip and an ELISA kit were developed to detect H7N9 virus,with the detection limit of 2¹ and 2^-2HAU/100u L for live H7N9 virus?A/Zhejiang/DTID-ZJU01/2013?,respectively.They are specific for detecting H7N9 virus and have potential application value.Part III.Preventive and therapeutic effects of mAbs on H7N9 virus infected mice.Mice were infected with 5×median lethal dose of highly pathogenic?HP?H7N9 virus,and then administered with a single dose of 10mg/kg of m Ab.MAbs 1H10 and 2D1showed protective effect on mice by neutralization and antibody-dependent cell-mediated cytotoxic effects,respectively.Thus two mAbs,1H10 and 2D1,have protective effects on low pathogenicity and HP H7N9 virus.To summarise,this study draws the following conclusions:?1?Six mAbs were developed in this study,all of which can specifically recognize the H7N9 virus and showed strong affinity.?2?Based on the mAbs?2D5 and 2F5?,a colloidal gold test strip and an ELISA kit for detecting H7N9 virus were developed and provided technical supports for H7N9prevention and control.?3?The mAbs?1H10 and 2D1?have significant preventive and therapeutic effects on H7N9 infected mice,suggesting 1H10 and 2D1 have good clinical application prospects.