Preparation Of The Monoclonal Antibodies Against EV71 VP1 And The VP1 Epitope Analysis

Enterovirus infection can cause hand,foot and mouth disease(HFMD),which is an acute infectious disease and mainly occurs in infants.Clinically,the younger patients are more serious,and the clinical symptoms happened quickily,obvious aggravation trend of the disease and unsatisfactory recovery often occur.At present,HFMD has posed a serious threat to the health and even life safety of infants and children.Enterovirus 71(EV71),as one of the main pathogens of HFMD,is highly infectious,which can lead to a high incidence of HFMD.Severe symptoms may show neurological symptoms and deaths.At present,common detection methods of EV71 include cell inoculation,lactating mice inoculation,serum neutralization test and RT-PCR.These methods are costly,time-consuming and it is often prone to false positive,which brings some troubles to early clinical diagnosis.Therefore,the establishment of a fast,accurate and easy to operate detection method has become the current EV71 diagnostic work of the urgent requirement.This experiment combined the advantages of monoclonal antibodies and ELISA to prepare EV71 VP1 monoclonal antibodies,and on this basis,a sandwich ELISA method of EV71 dual antibody was established.Two strains of EV71 VP1 monoclonal antibodies,named monoclonal CL-I and monoclonal CL-II,were obtained by cell fusion and three subclonal screening using the eukaryotic expression vector EV71 pcdna3.1-vp1 plasmid as immune antigen.In this study,the bioinformatics analysis of EV71 VP1 gene with online analysis software showed that the EV71 VP1 ORF gene had a total length of 891 nucleotides and a total encoding of 297 amino acids.The protein is acidic.Blast analysis revealed that the amino acid sequence of EV71 VP1 was 100% homologous with Nagoya(AB747373.1),which indicated that the EV71 VP1 gene obtained in this study was typical representative,and that the amino acid sequence of EV71 VP1 was 99.8% and 99% homologous with Nagoya(AB482183.1)and 16949(AB524109.1).In this study,a self-made EV71 monoclonal antibody CL-II which was used as the coated antibody to establish a sandwich ELISA method for EV71 dual antibodies and optimize its reaction conditions.At the same time,25 clinical EV71 blood samples and 24 serum samples of healthy children were detected,and the virus isolation and RT-PCR detection methods were compared.The coincidence rate of the three methods was high.The results showed that the sandwich ELISA method of EV71 was stable and could be used for rapid clinical detection of EV71.This study has laid a theoretical foundation for obtaining excellent clinical diagnostic reagents for prevention and treatment of HFMD.It is of great significance for grassroots laboratories to carry out early diagnosis of EV71,which involves the healthy growth of infants and children and human development,and has a broad development prospect and huge market demand.