Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa cells by transfection with the expression vector encoding cDNA of the LASV-NP gene. An immunoglobulin G enzymelinked immunosorbent assay (ELISA) using LASV-rNP and an indirect immunofluorescence assay using LASV-rNP-expressing HeLa cells were confirmed to have high sensitivity and specificity in the detection of LASV-antibodies. A novel monoclonal antibody to LASV-rNP, monoclonal antibody 4A5, was established. A sandwich antigen capture (Ag-capture) ELISA using the monoclonal antibody and an anti-LASV-rNP rabbit serum as capture and detection antibodies, respectively, was then developed. Authentic LASV nucleoprotein in serum samples collected from hamsters experimentally infected with LASV was detected by the Ag-capture ELISA. The Ag-capture ELISA specifically detected LASV-rNP but not the rNPs of lymphocytic choriomeningitis virus or Junin virus. The sensitivity of the Ag-capture ELISA in detecting LASV antigens was comparable to that of reverse transcription-PCR in detecting LASV RNA. These LASV rNP-based diagnostics were confirmed to be useful in the diagnosis of LF even in institutes without a high containment laboratory, since the antigens can be prepared without manipulation of the infectious viruses.Lassa fever (LF) is a viral hemorrhagic fever caused by Lassa virus (LASV), an Old World arenavirus. Many cases of LF occur in western Africa in countries such as Guinea, Sierra Leone, and Nigeria (7,23,27,(29)(30)(31). It is thought that LASV infects tens of thousands of humans annually and causes hundreds to thousands of deaths (34). Humans become infected through contact with infected excreta, tissue, or blood from the peridomestic rodent, Mastomys natalensis, the reservoir host of LASV (34). LASV can be transmitted to other humans via mucosal or cutaneous contact or through nosocomial contamination (27). More than 20 imported cases of LF have been reported outside the endemic region in areas such as the United States, Canada, Europe, and Japan (1,2,13,15,18,24,25). Recently, the potential for the use of hemorrhagic fever viruses, including LASV, as a biological weapon has been emphasized (5, 6). Therefore, the development of diagnostic systems for LF is important even in countries free from LF outbreaks to date.Manipulation of infectious LASV is necessary for the detection of specific antibodies. However, a high-containment laboratory (biosafety level 4 [BSL-4]) is required for handling infectious LASV and, therefore, the preparation of LASV antigens cannot be implemented in institutes without a BSL-4 facility. Within th...