title: A duplex recombinant viral nucleoprotein microbead immunoassay for simultaneous detection of seroresponses to human respiratory syncytial virus and metapneumovirus infections.
abstract: Serologic diagnosis of human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) infections has been shown to complement virus detection methods in epidemiologic studies. Enzyme immunoassays (EIAs) using cultured virus lysate antigens are often used to diagnose infection by demonstration of a ≥4-fold rises in antibody titer between acute and convalescent serum pairs. In this study, hRSV and hMPV nucleocapsid (recN) proteins were expressed in a baculovirus system and their performance compared with virus culture lysate antigen in EIAs using paired serum specimens collected from symptomatic children. The recN proteins were also used to develop a duplex assay based on the Luminex microbead-based suspension array technology, where diagnostic rises in antibody levels could be determined simultaneously at a single serum dilution. Antibody levels measured by the recN and viral lysate EIAs correlated moderately (hRSV, r(2)=0.72; hMPV, r(2)=0.76); the recN EIAs identified correctly 35 of 37 (94.6%) and 48 of 50 (96%) serum pairs showing diagnostic antibody rises by viral lysate EIAs. Purified recN proteins were then coupled to microbeads and serum pairs were tested at a single dilution on a Luminex MAGPIX(®) analyzer. The duplex recN assay identified correctly 33 of 39 (85%) and 41 of 47 (86.7%) serum pairs showing diagnostic rises to hRSV and hMPV, respectively. The recN assay permits simultaneous testing for acute hRSV and hMPV infections and offers a platform for expanded multiplexing of other respiratory virus assays.
authors.affiliation: Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States; Battelle, Atlanta, GA, United States.
authors.forename: Yange
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: International Centre for Diarrheal Diseases Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh.
authors.forename: W Abdullah
authors.initials: WA
authors.lastname: Brooks
authors.affiliation: International Centre for Diarrheal Diseases Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh.
authors.forename: Doli
authors.initials: D
authors.lastname: Goswami
authors.affiliation: International Centre for Diarrheal Diseases Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh.
authors.forename: Mustafizur
authors.initials: M
authors.lastname: Rahman
authors.affiliation: Woods Institute of the Environment, Stanford University, Stanford, CA, United States.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Luby
authors.affiliation: Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States. Electronic address: dde1@cdc.gov.
authors.forename: Dean D
authors.initials: DD
authors.lastname: Erdman
mesh_terms: D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D002675:Child, Preschool; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D007223:Infant; D029121:Metapneumovirus / Q000235:genetics / Q000276:immunology*; D008863:Microspheres; D009698:Nucleoproteins / Q000235:genetics; D018184:Paramyxoviridae Infections / Q000175:diagnosis* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis* / Q000276:immunology; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D009698:Nucleoproteins; D011993:Recombinant Fusion Proteins
keywords: Human metapneumovirus (hMPV); Human respiratory syncytial virus (hRSV); Immunoassay; Serology