Development of recombinant nucleoprotein-based diagnostic systems for Lassa fever.
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.
Saijo
Masayuki
M
Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. msaijo@nih.go.jp
Georges-Courbot
Marie-Claude
MC
Marianneau
Philippe
P
Romanowski
Victor
V
Fukushi
Shuetsu
S
Mizutani
Tetsuya
T
Georges
Alain-Jean
AJ
Kurata
Takeshi
T
Kurane
Ichiro
I
Morikawa
Shigeru
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007313:Insecta; D007835:Lassa Fever / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D018985:Epitopes, B-Lymphocyte; D007074:Immunoglobulin G; D009698:Nucleoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins