Development Of The Time-resolved Fluoroimmunoassay For Detection Of E Protein Of Japanese Encephalitis Virus And Recombinant L1 Protein Of Human Papillomavirus Type 68

Japanese encephalitis(JE)is a zoonotic disease with a high mortality rate in children and the elderly in many Asian countries.Close to 3 billion people worldwide are believed to be at risk of JE,and approximately 20,000 clinical cases resulting in 6000 deaths are reported each year.Of these cases,about 25-30%are fatal and 50%result in permanent neuropsychiatric sequelae,however only one third of patients recover completely.Since the introduction of JE vaccines,their efficacy testing has been a complex process.PRNT is the most commonly used method to test JE vaccine efficacy.It is the most reliable,sensitive,and specific assay,and it compares the amount of vaccine required to induce neutralizing antibodies in mice with the amount of a reference vaccine preparation required to produce the same effect.But the process is lengthy,laborious and technically demanding.Testing requires a large number of experimental animals as well as trained staff.In the first two chapters of this study,a time-resolved fluoroimmunoassay for detection of the E protein of inactivated JE vaccine was developed.1.Expression and pairing screening of monoclonal antibodies;2.Establishment of methodology;3.Evaluate the performance indexes of the kit,including sensitivity,precision,accuracy and specificity.The effectiveness and necessity of human papillomavirus(HPV)vaccination to prevent HPV infection and cervical cancer are increasingly recognized by the public.Much attention has been focused on the 15-valent HPV vaccine,which protects against almost all high-risk types of HPV as defined by WHO.However,with the continuous improvement of vaccine potency,the quality control of HPV vaccine production process faces more challenges.HPV type 68 virus-like particles(VLPs)are one of the unique components that distinguish the 15-valent HPV vaccine from existing vaccines.The precise quality control of VLPs is a new requirement for vaccine manufacturers.In this study,a novel time-resolved fluoroimmunoassay(TRFIA)was established for rapid and accurate automatic quality control of E protein in inactivated JE vaccine and HPV68 VLPs in HPV vaccine.Two pairs of murine monoclonal antibodies specifically targeting JE E protein or HPV68 L1 protein were used to establish the classical sandwich detection method.The linear range of JE E protein time-resolved immunoassay kit was 0.78125 IU/ml-25 IU/mL,the sensitivity was 0.01 IU/mL,the intra-assay coefficient of variation was less than 5%,and the inter-assay coefficient of variation was less than 10%.The dilution recovery was between 90%and 110%.The HPV68 VLPs time-resolved immunoassay kit had a linear range of 1000 ng/mL,a sensitivity of 0.08 ng/mL,and both intra-and inter-assay coefficients of variation were less than 10%.The dilution recovery ranged from 90%to 110%.Except for the pretreatment of vaccine samples,the entire analytical process was completed by fully automatic machines,which saved detection time and avoided manual errors.The results showed that the two novel TRFIA could efficiently and reliably analyze JE vaccine titer and HPV68 VLPs.The novel TRFIA has the advantages of speed,durability,high sensitivity,high accuracy,wide detection range and good specificity.It is expected to provide a new detection method for the quality control of various types of VLPs.In conclusion,the novel TRFIA has important application value in the quality control of JE vaccine and HPV vaccine.