title: Development of a quantitative enzyme linked immunosorbent assay for monitoring the Enterovirus 71 vaccine manufacturing process.
abstract: Enterovirus 71 (EV71), the etiologic agent causes outbreaks with significant mortality in young children in Asia and currently there is no vaccine available. In this study, we report a quantitative enzyme linked immunosorbent assay (Q-ELISA) to determine the concentration of the EV71 VP2 antigen. EV71 virus-like particles (VLPs) were produced in the baculovirus expression system and used as the EV71 antigen reference standard. Antisera from both EV71-immunized chickens and rabbits were very efficient and useful as capture antibodies to bind various forms of EV71 antigens, whereas a commercial VP2-specific virus neutralizing monoclonal antibody MAB979 was found to be suitable for quantifying the amount of VP2 antigen. This Q-ELISA was used successfully to determine VP2 content at each stage of EV71 vaccine manufacturing process, particularly during the upstream harvest, downstream purification and viral inactivation steps. The amount of VP2 antigen and the magnitude of neutralizing titers were found to be dose-dependent in mice immunized with vaccine candidates. These results indicate that Q-ELISA could provide off-line timely quantitative measurements of VP2 antigen throughout the production cycle to evaluate critical attributes and conditions that may affect virus yields in culture media, the quality of purification methods, the stability and potency of final vaccine formulations.
authors.affiliation: Vaccine R&D Center, National Health Research Institutes, Zhunan Town, Miaoli County 350, Taiwan.
authors.forename: Chia-Chyi
authors.initials: CC
authors.lastname: Liu
authors.forename: Hsuen-Wen
authors.initials: HW
authors.lastname: Chang
authors.forename: Grace
authors.initials: G
authors.lastname: Yang
authors.forename: Jen-Ron
authors.initials: JR
authors.lastname: Chiang
authors.forename: Yen-Hung
authors.initials: YH
authors.lastname: Chow
authors.forename: I-Hsi
authors.initials: IH
authors.lastname: Sai
authors.forename: Jui-Yuan
authors.initials: JY
authors.lastname: Chang
authors.forename: Sue-Chen
authors.initials: SC
authors.lastname: Lin
authors.forename: Charles
authors.initials: C
authors.lastname: Sia
authors.forename: Chia-Hsin
authors.initials: CH
authors.lastname: Hsiao
authors.forename: Ai-Hsiang
authors.initials: AH
authors.lastname: Chou
authors.forename: Pele
authors.initials: P
authors.lastname: Chong
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology*; D000956:Antigens, Viral / Q000032:analysis* / Q000276:immunology; D001709:Biotechnology / Q000379:methods; D036022:Capsid Proteins / Q000032:analysis* / Q000276:immunology; D002522:Chlorocebus aethiops; D004770:Enterovirus / Q000254:growth & development / Q000276:immunology*; D004769:Enterovirus Infections / Q000276:immunology / Q000517:prevention & control; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014611:Vaccination; D014709:Vero Cells; D014765:Viral Vaccines / Q000592:standards
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D014765:Viral Vaccines