Summary. -Enterovirus 70 (EV70) is the causative agent of acute hemorrhagic conjunctivitis (AHC), for which no effective vaccine is available. This study revealed a high reactivity of the N-terminal region of EV70 VP1 (VP1-1) with an anti-EV70 mouse serum. The analysis of overlapping synthetic peptides of VP1-1 identified a B-cell epitope in this region. The E-peptide (14-ANTVESEIKAELGVI-28) showing the highest reactivity with the anti-EV70 serum induced neutralizing antibodies in mice and reduced the virus titer in the eyes, suggesting that it is a candidate vaccine against AHC caused by EV70.Keywords: enterovirus 70; AHC; B-cell epitope; peptide vaccine * Corresponding author. E-mail: jhnam@catholic.ac.kr; phone: +82-2-2164-4917. # These authors contributed equally to this work. Abbreviations: EV70 = enterovirus 70; AHC = acute hemorrhagic conjunctivitis; VP1-1 = virus capsid protein VP1 amino-terminus; VP1-2 = virus capsid protein VP1 middle; VP1-3 = virus capsid protein carboxy-terminus

Japanese encephalitis virus (JEV) causes diseases that attack the human central nervous system. Traditionally, the quality control for JEV vaccines, in which the plaque reduction neutralization (PRN) titer is measured by the national control laboratories before the vaccine batches are marketed, has required laboratory animal testing. However, classical animal tests have inherent problems, including the very fact that animals are used, ethical issues, and the possibility of error. In this study, JEV antigen was measured in an in vitro assay to assess the feasibility of replacing in vivo assays that measure the PRN titers of JEV vaccines. We constructed a double‐sandwich enzyme‐linked immunosorbent assay (DS‐ELISA) that could detect JEV envelope (E). Initially, monoclonal antibodies (mAbs) directed against the JEV E protein were generated and characterized. We isolated 18 mAbs against JEV E protein, and most were the IgG1 or IgG2a isotype. The mAbs (5F15 and 7D71) were selected as the most suitable mAb pair to detect JEV E protein. DS‐ELISA with this pair detected as little as approximately 3 μg/mL JEV E protein and demonstrated a relationship between the amount of JEV E protein and the PRN titer. From these results, we surmise that this DS‐ELISA may be useful, not only in terms of measuring the amount of JEV E protein, but also as a substitute for the PRN test for JEV vaccine evaluation.