Recombinant Japanese encephalitis (JE) virus proteins were evaluated as antigens for serodiagnosis of JE using an enzyme-linked immunosorbent assay (ELISA). The premembranel membrane (prMiM) and envelope (E) proteins of JE virus were expressed in HeLa cells infected with a recombinant vaccinia virus that encodes the JE virus prM and E genes and were released from cells in a particulate form. The particulate antigens were partially purified from culture fluid from the infected cells by precipitation of particles with polyethylene glycol and then dissociated from the particles with 0.1% Triton X-100. This antigen preparation was used to evaluate one preimmune and two postvaccination sera from 20 volunteers given three inoculations of the commercial JE vaccine (Biken vaccine) by a conventional ELISA. The results from this assay correlated with neutralization data. The results of an IgM capture ELISA carried out with the recombinant antigen also correlated with the results of an existing IgM capture ELISA performed with JE virus-infected mouse brain, when tested with 29 serum and 13 cerebrospinal fluid samples from JE patients. These results indicated that recombinant JE virus antigens are useful for ELISA as an antigenically equivalent, highly productive, and safe alternative to authentic JE virus antigens. 1 0 1996 Wiley-Liss, Inc.