Development Of A Sandwich ELISA For Detection Of CEV Antigen And Mapping Of The Antigen Epitope For Cev-encoded VP1

The genus Enterovirus within the family of Picornaviridae consists of 9 species(A,B,C,D,E,F,G,H and J)of Enteroviruses(EV)and 3 species(A,B,C)of Rhinoviruses based on the latest virus taxonomy.Those viruses are the etiological agents contributing to neurological,respiratory and digestive disorders in human and animals.Out of 9 Enterovirus species,Enterovirus E(EV-E),Enterovirus F(EV-F)and Enterovirus G(EV-G)are closely related to diseases affecting on the livestock industry,where EV-E and EV-F(formerly named bovine enterovirus A and B)are the causative agents of enterovirus infections in cattle.The EV-G(formerly named porcine enterovirus B)is the causative agents of swine enterovirus infections.Enterovirus infection in sheep/goat is an emerging infectious disease that was initially reported in 2012 by Hungary.In 2014,our laboratory isolated an enterovirus strain(designated as CEV-JL14)from a goat herd demonstrating severe watery diarrhea with the morbidity and mortality up to 50-80%,respectively.Currently,it remains largely unknown related to the viral pathogenesis,the diagnosis,prevention and control this emerging infection.To establish a specific,sensitive,rapid and simple method for the detection of CEV antigen and to provide the technical means for the epidemiological study,we performed RT-PCR to amplify the VP1 gene encoded by CEV-JL14.The amplified VP1 gene was cloned to prokaryotic expression vector to generate recombinant plasmid.After induction with IPTG,the recombinant GST-fusion VP1 protein was expressed,purified and used to generate the monoclonal antibody and rabbit polyclonal antibody against VP1.A sandwich ELISA was developed for detecting caprine/ovine enterovirus antigens using the antibodies generated and ELISA kit for CEV antigen detection was assembled and evaluated.The results demonstrated that the established sandwich ELISA and ELISA kit were specific,sensitive,fast,easy and sample for detection of CEV antigen.The ELISA kit was stable at least for 180 days kept at 4℃.Epidemiological investigation of CEVinfection from sheep/goat herds in Jilin Province and Inner Mongolia autonomous regions showed that enterovirus infection existed in different herds among these areas with the infection rate up to 11.4%-100.0%.Furthermore,the epitope of the monoclonal 6A1 was dissected by peptide scanning technique,and the antigenic epitope of the monoclonal antibody 6A1 was mapped to be 152TPPTDQDTYQWQT164,thus laying a solid basis for future study related to viral pathogenesis and immunology for caprine enterovirus infection.In summary,a monoclonal antibody and polyclonal antibody against CEV-VP1 was prepared and used to establish the sandwich ELISA for detection of CEV antigen.The established ELISA was specific,sensitive,fast,and easy for epidemiological investigation and diagnosis for CEV infection.The findings in this study showed that CEV infections were widely detected among sheep/goat herds in different regions.At the same time,the specific epitope of monoclonal antibody 6A1 was mapped.The research in this study will provide a sensitive,specific,simple and rapid antigen detection method for diagnosis and epidemiologic survey for CEV infection,and an invaluable data for prevention and control of this emerging infectious diseases.