title: Development of an enzyme-linked immunosorbent assay-based test with a cocktail of nucleocapsid and spike proteins for detection of severe acute respiratory syndrome-associated coronavirus-specific antibody.
abstract: A new enzyme-linked immunosorbent assay (ELISA)-based immunoglobulin G (IgG)-plus-IgM antibody detection test for severe acute respiratory syndrome (SARS) has been developed by using a cocktail of four recombinant polypeptides as the antigen. These recombinant fragments were designed as parts of two different structural proteins from SARS-associated coronavirus (SARS-CoV). One recombinant polypeptide, S251-683, was designed as part of the spike glycoprotein, and the other three polypeptides comprised almost the whole nucleocapsid protein, avoiding the last 25 C-terminal amino acids. Immunization with a cocktail of these four polypeptides yielded a specific polyclonal antibody that is able to recognize SARS-CoV-infected cells by an immunofluorescence assay. This polypeptide cocktail was also used to set up an ELISA-based IgG-plus-IgM antibody detection test, which showed 99% specificity and 90% sensitivity upon evaluation using sera from 100 healthy negative controls and 20 SARS patients. Separate immunoreactivity assays with each recombinant polypeptide demonstrated that a combination of N and S protein fragments was more suitable than the individual peptides for developing a serological assay for SARS-CoV.
authors.affiliation: Laboratorios Vircell, SL, Granada, Spain.
authors.forename: Luis G
authors.initials: LG
authors.lastname: Giménez
authors.forename: Jose
authors.initials: J
authors.lastname: Rojas
authors.forename: Almudena
authors.initials: A
authors.lastname: Rojas
authors.forename: Joaquín
authors.initials: J
authors.lastname: Mendoza
authors.forename: Ana G
authors.initials: AG
authors.lastname: Camacho
mesh_terms: D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D008562:Membrane Glycoproteins / Q000276:immunology; D019590:Nucleocapsid Proteins / Q000276:immunology; D011994:Recombinant Proteins; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D008562:Membrane Glycoproteins; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus