Preparation And Identification Of Anti-EV71 VP2 Monoclonal Antibody

Background:Enterovirus 71 (EV71) is the major cause of hand-foot-and-mouth disease (HFMD). EV71 infection can be accompanied by a series of syndromes with or without central nervous system involvement, including herpangina, aseptic meningitis, poliomyelitis-like paralysis and possibly fatal encephalitis. Since it was initially identified in California in 1969,12 large scale outbreaks have been reported worldwide, including in Australia, Southeast-Asia and Europe. However, the most severe outbreak with the highest mortality occurred in Asia. The capsid proteins of EV71 play an important role in EV71 infection and induction of immune response. Recently, considerable efforts have been made in the fields concerning the viral capsid proteins of EV71 and the corresponding monoclonal antibodies.Objective:Synthetic peptides based on VP2 of EV71 were used to prepare anti-EV71 capsid protein VP2 monoclonal antibody. The target antibody would be a basis to produce diagnosis kit for EV71 infection and preparation of EV71 vaccine in the future. Methods:Based on the GenBank sequence and reference, VP2 peptide amino acid sequences were decided by use of computer-aided design. The peptides were synthetized and conjugated with Keyhole Limpet Hemocyanin (KLH) to heighten immunogenicity of antigen. BALB/c mice were immunized with the synthetized peptides to produce anti-EV71 VP2 monoclonal antibody. Direct ELISA assay was used to detect the monoclonal antibodies and identify the level of antibodies. The generated mAbs containting ascetic fluid was purified by affinity chromatograph. The specificity of the anti-EV71 VP2 monoclonal antibody was confirmed by immune cells staining, western blot and immuno-precipitation against EV71 virus.Results:Four hybridoma cell lines were obtained by hybridoma technique. Isotype of these four monoclonal antibidies (mAbs) were all IgG1. The titres of the four mAbs measured by ELISA were 1×105-1×106. Immunocytochemical staining showed that anti-EV71 VP2 mAbs could specificly bind to strains of EV71. Western blot and immuno-precipitation showed the produced mAbs specificly reacted to the lysate of EV71 strain isolated from clinical. It showed a clear band in molecular weight of about 40KD, we considered the antibodies could identify the specific peptide fragments of EV71 virus, showing good specificity of the produced monoclonal antibodies.Conclusion:By ELISA method, immunocytochemical staining, western-blot and immuno-precipitation, the anti-EV71 VP2 antibodies were able to sensitively and specificly identify EV71 capsid protein. It indicated that anti-EV71 VP2 monoclonal antibodies were successfully prepared in this study. The antibodies can be used in a wide range of applications, such as the diagnosis and treatment of EV71 infection and vaccine production, research on the structure and function of the EV71, and antibody analysis.