Recombinant protein-based ELISA and immuno-cytochemical assay for the diagnosis of SARS.
A new Coronavirus (SARS-CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in-house ELISA based on SARS-CoV recombinant antigens was developed. The SARS-CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine-tagged proteins. Six reference sera from SARS patients were used to detect virus-specific IgG in an ELISA using each recombinant protein as coating antigen. High-titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non-SARS, low-tract respiratory infections. Specific hyper-immune sera to SARS-CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS-CoV infected and uninfected cells.
Carattoli
Alessandra
A
Department of Infectious, Parasitic and Immuno-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
Di Bonito
Paola
P
Grasso
Felicia
F
Giorgi
Colomba
C
Blasi
Francesco
F
Niedrig
Matthias
M
Cassone
Antonio
A
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics; D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis*; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology
D000914:Antibodies, Viral; D000956:Antigens, Viral; D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D007106:Immune Sera; D007074:Immunoglobulin G; C000706237:M protein, SARS-CoV; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins