Establishment And Preliminary Application Of A Novel Neutralization Assay For Coxsackie Virus B 1

Since the large-scale outbreak of hand foot and mouth disease(HFMD)in fuyang city,AnHui province,occurred in 2008,over 20 million cases have reported by the end of 2018,among which about 3,600 cases have died.The number of cases and deaths ranked first among class C infectious diseases in China,which seriously endangering public health.There are more than 20 human enteroviruses leading to HFMD.In addition to the main pathogens enterovirus 71(EV71)and coxsackievirus A16(CVA16),coxsackievirus B1(CVB1)is also an important pathogen causing HFMD.Studies have shown that CVB1 infection can also cause aseptic meningitis,myocarditis,meningocephalitis and associated with type 1 diabetes.However,there is no prevention or treatment for CVB1,and it is urgent to carry out basic and applied research on related prevention and treatment methods.In order to further establish and improve relevant research techniques and methods,this study established a rapid and efficient noval neutralizing antibody detection method for CVB1(Nt-ELISPOT)based on ELISPOT,providing technical support for the development of CVB1 vaccine and drug and the study of serum epidemiology.In this study,the CVB1 clinical isolate XM0108 strain isolated from Quanzhou,Fujian Province.The evolution of the nucleic acid sequence showed that the strain was highly similar to the reported CVB1 strain in China and belonged to the main epidemic strain.The CVB1 virus cultured and purification conditions of virus particle have had been explored.A stable and efficient virus culture and preparation method was established.The method of concentrating and purifying the virus stock solution was established.Then,a CVB1 virus particle having a higher purity been obtained by a sucrose density centrifugation method.Obtaining monoclonal antibodies with specific and good reactivity to CVB1 virus is an important basis for establishing a new immunoassay based on ELISPOT technology.In this study,mice were immunized by inactivated virus particles by intraperitoneal injection,and 6 monoclonal antibodies against CVB1 were screened by indirect ELISA.By analyzing the binding activity and reaction specificity of the relevant monoclonal antibodies against CVB1 virus,2E6 monoclonal antibody with better reactivity was been selected as the detection antibody of Nt-ELISPOT detection method.Furthermore,through the system to explore the Nt-ELISPOT detection method in the detection of monoclonal antibody dilution ratio,virus infection dose and culture time,this study based on ELISPOT technology to establish a rapid and high-flux CVB1 neutralization test method.Compared with the traditional cytopathic effect(CPE)-based neutralization test method(Nt-CPE),this method can significantly shorten the detection time(from 5-7 days to 1 day),and the detection results of the two methods are good.consistency.In this study,the Nt-ELISPOT method was used to screen the CVB1 neutralizing monoclonal antibody,and 6 CVB1 neutralizing monoclonal antibodies were obtained.Among them,5 monoclonal antibody subtypes are IgG2a,and one strain is IgGl subtype.Neutralization ability analysis showed that 8A10 had relatively better neutralizing activity.In this study,8A10 and CVB1 immune antiserum was further been evaluated by the neonatal mouse infection model.The results showed that 8A10 and CVB1 immune antiserum could effectively protect mice from lethal challenge of CVB1 by passive immunization.At the same time,this study applied these novel neutralization test assay to detect CVB1 neutralizing antibody levels in serum samples from 515 healthy people in Xiamen.The results showed that the positive rate of CVB1 neutralizing antibody in the above serum samples was 27%,and the positive rate of serum neutralizing antibody in the 20-39 age group was the highest,suggesting that there was a previous epidemic infection of CVB1 in Xiamen.In summary,this study established a novel CVB1 neutralizing antibody detection method.It can applied with quickly,efficiently and in high-throughput to the screening and evaluation of CVB1 neutralizing antibodies and related sero-epidemiological investigations,which can provide important support for the research of CVB1 prevention and treatment methods.