RESUMEN
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.
Asunto(s)
Fiebre de Lassa/diagnóstico , Nucleoproteínas/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Antígenos Virales/inmunología , Baculoviridae/genética , Cricetinae , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos de Linfocito B/inmunología , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Haplorrinos , Células HeLa , Humanos , Inmunoglobulina G/inmunología , Insectos , Fiebre de Lassa/genética , Fiebre de Lassa/inmunología , Fiebre de Lassa/virología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Nucleoproteínas/biosíntesis , Nucleoproteínas/genética , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
There have recently been large outbreaks of Marburg hemorrhagic fever (MHF) caused by Marburgvirus (MARV) in the Democratic Republic of Congo and Angola. The development of reliable diagnostic systems for MHF is urgently needed. An antigen-capture enzyme-linked immunosorbent assay (Ag-capture ELISA) using either of the two monoclonal antibodies (2A7 and 2H6) produced by immunizing mice with recombinant nucleoprotein of MARV was described (Journal of Medical Virology, 76, 111-118, 2005). In the present study, it was revealed that the Ag-capture ELISA specifically detected authentic MARV antigen and that the sensitivity of the Ag-capture ELISA was at a level similar to that of reverse-transcription polymerase chain reaction. These results suggest that the Ag-capture ELISA using the monoclonal antibodies, 2A7 and 2H6, is applicable to the diagnosis of MHF.