Development And Identification Of Monoclonal Antibody Specific For Citrullinated ?-enolase

?-Enolase(ENO1)is a key glycolytic enzyme,which catalyze the conversion of PGA with PEP in glycolysis.In addition to its innate glycolytic function,?-enolase also plays an important role in several biological and pathophysiological processes,its ability to serve as plasminogen receptor on the surface of cells suggest that it may plays an important role in the intravascular and pericellular fibrinolytic system,facilitate cell communication;serve as c-myc promoter-binding protein 1(MBP-1)indicate that it can bind to the c-myc p2 promoter and negatively regulates transcription of the protooncogene;and also identified as autoantigen inducing autoimmune response.Recent accumulation of evidence revealed that citrullination of ?-enolase has been implicated in numerous diseases,including Neurodegenerative disease,autoimmune disorders and bacterial infection and so on,especially Alzheimer's disease and Rheumatoid arthritis.Therefore,antibody could specifically bind to citrullinated ?-enolase will be urgently need for further study its role in pathogenesis of related complex diseases.Bioinformatic analysis of ?-Enolase B cell epitope showing that the immunodominant epitope was loacted at 9R-25F,two arginine in the 9R-25F region was substituted by citrulline before sythesis and named citrullinated ?-enolase peptide(CEP).CEP-BSA artificial antigen was prepared by novel coupling method.CEP-BSA could inducing specific immune response in BALB/c mouse.By exploiting hybridoma technique to generate Monoclonal antibody specific for citrullinated ?-enolase,New monoclonal antibody's specificity were verified by WB and IHC.Results are as follows:1)Bioinformatic analysis of ?-Enolase B cell epitope showing that the immunodominant epitope was loacted at 9R-25F,two arginine in the 9R-25F region was substituted by citrulline before sythesis,named citrullinated ?-enolase peptide(CEP).CEP-BSA artificial antigen was prepared by novel coupling method and was identified by SDS-PAGE.2)CEP-BSA artificial antigen was performed by standard bi-weekly immunization,then titer in serum shows the immunization was successful and ready for cell fusion.Celrl fusion was performed by PEG,followed with subcloning(limiting dilution method),We screened out one strain of hybridoma that secrete CEP specific monoclonal antibody constantly,namely 9B5,subtype is IgG2b.3)9B5 monoclonal antibody was purified by Ammonium Sulfate Precipitation combine with Caprylic Acid Precipitation.The result of Western blotting showed that 9B5 monoclonal antibody could specifically bind to citrullinated ?-enolase.The result of IHC demonstrated that 9B5 monoclonal antibody could specifically bind to citrullinated?-enolase expressed on brain cell of aging animal model.New mouse monoclonal antibody specific for CEP developed successfully in this research,It would aid in the further study of CE's role in pathogenesis of related complex diseases.