Development Of Antigen Detection Method For Recombinant Human Enterovirus 71 Virus-like Particle Vaccine

Human Enterovirus71(HEV71)is one of the main pathogens that can cause human Hand,foot and mouth disease(HFMD).The susceptible group is children under 5 years old,some of them will have severe respiratory difficulties,accompanied by complications of nervous system and circulatory system,and even death.At present,there is no symptomatic treatment for EV71-induced hand,foot and mouth disease,the usual treatment is mainly supportive treatment,and preventive vaccine is an effective means to prevent EV71 infection.At present,there are three kinds of EV71 inactivated vaccines on the market in China,but no virus-like particle(VLPs)vaccine has been released.Because of the unique safety and immunogenicity of VLPs and the need to develop EV71-related combined vaccine,EV71 VLPs vaccine has become a hot spot in research and development.The aim of this study was to establish a method for the detection of EV71VLPs(IB-EV71 VLPs)prepared by insect cell-baculovirus expression system in order to meet the requirements of the detection of antigen content in the development of IB-EV71 VLPs vaccine.Japanese white rabbits were immunized with IB-EV71 VLPs to obtain a serum with antibody titer greater than 1:100000 and purified by affinity chromatography to obtain polyclonal antibodies.A double-antibody sandwich ELISA method was established to detect IB-EV71 VLPs using polyclonal antibody and HRP-labeled anti-EV71P1 antibody.The optimal coating concentration and dilution ratio of HRP-labeled antibody were screened.The specificity,linearity,range,accuracy,limit of quantification and precision of the method were evaluated according to the requirements of method verification.The results show that the established method has good specificity.In the range of 25-400ng/ml,the linear relationship was good,and the R2 value was greater than 0.99.The recoveries within the detection range were between 86% and 116%.The limit of quantitation was 2ng/ml.The relative standard deviations(RSD)of both intraplate precision and inter-plate precision were less than 15%.In summary,this study established a double-antibody sandwich ELISA method to detect IB-EV71 VLPs.The method has good specificity,linearity,range,accuracy and precision,and the limit of quantification is 2ng/ml,which meets the basic requirements of the guiding principles of bioanalytical methodology validation,and lays a foundation for the development and quality control of IB-EV71 VLPs vaccine.