Study Of Broad-spectrum Detecting Monoclonal Antibodies And Novel Universal Neutralization For Human Enterovirus

Recent years,the large-scale outbreak of hand foot and mouth disease(HFMD)in the world,especially in South Asia including our country re-attracted people’s attention to enterovirus.Researchers’ attention mainly focused on EV71 and CVA16 which are the major pathogens of HFMD in Asia.EV71 vaccine is currently in clinical phase Ⅲ,and CVA16 vaccine is being developed.However,there is few researches of other pathgens of HFMD as CVA6 or CVA10.Therefore,it is very important to further establish and improve the techniques and methods for human enterovirus reseach.This study explored the construction of mAbs which have a broad-spectrum of recognition ability to human enterovirus,and developed a general nutralization assay.This study compared the amino acid sequences of 64 different serotypes of enterovirus to screen the relatively conservative polypeptide sequences.Then mice immunized with protein particles displayed by HbcAg C149 carriers which inserted with selected peptides.The results indicate that the mouse serum got from immunization of polypeptide epitope aal266-1281 in 2C protein has a broad-spectrum of affinity to human enterovirus,and could be successfully used in immunofluorescence assay.This study further screened monoclonal antibodies against the epitope aa1266-1281,and proved that the mAbs also have recognition ability to different serotypes or genotypes of enterovirus.At the same time,the mAb could also be successfully applied to immunohistochemical detection of mice model infected by enterovirus.The results show that the mAb obtained in this study has good application value,and provides an important tool in the study of basic research and clinical diagnosis for human enterovirus.This study further developed a quick,high-throughput and general neutralization assay for human enterovirus based on the mAb obtained.Prior to this study,our laboratory has been successfully established a EV71 enzyme-linked immunospot(ELIspot)neutralization assay,which could quick detect neutralizing antibody titers of serum samples by Elispot system.In this study,the assay was further optimized for human enterovirus versatility based on the broad-spectrum affinity mAb.This study evaluated several clinical serum by novel detection assay,and results showed that our assay has good parallel with the original one,preliminary indicated the reliability of the method.Thus,this method will be an important tool for the development of human enterovirus vaccine especially universal vaccine with a good prospect.