Preparation And Application Of Anti-H7 Subtype Influenza Virus Monoclonal Antibody

Objective:The human monoclonal antibody against H7 subtype influenza virus was prepared,and the genetic characteristics and functional activities of the antibody were analyzed.At the same time,the murine monoclonal antibody of anti-H7 subtype influenza virus was prepared,and the double antibody sandwich ELISA was used to rapidly detect the H7 subtype avian influenza virus,which provided an approach for the diagnosis and prevention of H7N9 virus.Methods:The blood samples of patients infected with H7N9 was collected,and the peripheral blood memory B cells were sorted,cultured and cloned.The light and heavy chain genes of the antibody were co-transfected into 293T cells,then the mAbs were screened and purified.The genetic characteristics and functional activities of fully human antibodies were initially identified.Hybridoma preparation technology was used to fuse the spleen cells of immunized mice and myeloma cells,and H7-specific monoclonal antibodies were screened.DAS-ELISA was used to detect H7 antigen,and its sensitivity and specificity were identified.Results:（1）Gene cloning and sequencing of 9 cultured clones,24 VH germline genes and 14 Vκgermline genes were obtained from 1#sample,whlie 17 VH germline genes and 12 Vκs germline genes were obtained from 3#sample.The heavy chain genes of 1#sample are mainly IGHV4-39,IGHV1-69,IGHV4-59 and IGHV3-13,while the light chain gene was mainly IGKV1-39.The heavy chain genes of 3#sample are mainly IGHV3-9,IGHV3-7,IGHV1-69 and IGHV1-2,while the light chain gene was mainly IGKV1-5.（2）Obtaining 5 human mAbs,wherein antibody 56B11-2/4 exhibits neuraminidase inhibition activity,and its IC₅₀0 is about 52.46μg/ml;10F10-13/13 is a broad-spectrum binding antibody with strong ELISA binding activity.（3）Twelve H7-specific murine mAbs were obtained,of which 5 strains possess HI activity.A DAS-ELISA was used to detect the H7 antigen using two H7-specific mAbs named 2B6 and 5E9.The results show that the established DAS-ELISA platform only reacts with the H7 subtype influenza virus and does not react with other subtype viruses.The sensitivity of DAS-ELISA was tested with H7 subtype influenza virus and recombinant H7 protein.The detection limit for live virus was 0.5 HAU/50μl for A/Guangdong/17SF003/2016（H7N9）,2HAU/50μl for A/Netherl and s/219/2003（H7N7） and A/Anhui/1/2013（H7N9）,respectively.For H7protein,it was dose-responding and the detection limit was as low as 10 ng/ml.Conclusion:（1）One strain human monoclonal antibody having a neuraminidase-inhibiting ability and another strain having a strong broad-spectrum binding antibody were obtained.（2）Two strains of mouse monoclonal H7-specific mAbs were used to establish a DAS-ELISA,which showed high sensitivity and strong specificity for H7 antigen detection,and could be used for virus detection and HA vaccine quantification.