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Clinical and Vaccine Immunology, September 2007, p. 1182-1189, Vol. 14, No. 9
1071-412X/07/$08.00+0     doi:10.1128/CVI.00101-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of Recombinant Nucleoprotein-Based Diagnostic Systems for Lassa Fever

Masayuki Saijo,^1*, Marie-Claude Georges-Courbot,² Philippe Marianneau,² Victor Romanowski,³ Shuetsu Fukushi,¹ Tetsuya Mizutani,¹ Alain-Jean Georges,⁴ Takeshi Kurata,⁵ Ichiro Kurane,¹ and Shigeru Morikawa^1,

Department of Virology 1, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan,¹ Unit of Biology of Viral Emerging Infections, Institute Pasteur, IFR 128 Biosciences Lyon-Gerland, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France,² Instituto de Bioquímica y Biologíca Molecular, Facultad de Ciencias Exactas, Universidad Nacional de a Plata, 1900 La Plata, Argentina,³ Laboratory P4 Jean-Merieux-INSERM, 21 Avenue Tony Garnier 69365 Lyon Cedex 07, France,⁴ Department of Pathology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-864, Japan⁵

Received 1 March 2007/ Returned for modification 22 April 2007/ Accepted 20 June 2007

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 enzyme-linked 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.

Published ahead of print on 18 July 2007.

M.S. and S.M. contributed equally to this study.