Junin virus (JUNV), Machupo virus, Guanarito virus, Sabia virus, and Chapare virus are members of New World arenavirus clade B and are the etiological agents of viral hemorrhagic fevers that occur in South America. In this study, we produced three monoclonal antibodies (MAbs) to the recombinant nucleocapsid protein of JUNV, designated C6-9, C11-12, and E4-2. The specificity of these MAbs was examined by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, and an epitope-mapping method. Using these MAbs, we developed antigen (Ag) capture ELISA systems. We showed that by using MAb C6-9, JUNV Ag was specifically detected. On the other hand, by using MAb C11-12 or E-4-2, the Ags of all human pathogenic South American arenaviruses were detected. The combined use of these Ag capture ELISA systems in the present study may be useful for the diagnosis of acute-phase viral hemorrhagic fever due to infection by a South American arenavirus.