title: Antigen detection in human respiratory Coronavirus infections by monoclonal time-resolved fluoroimmunoassay.
abstract: BACKGROUND
  The diagnosis of respiratory infections by detecting viral antigens has received considerable attention using immunofluorescent assays (IFA) and enzyme immunoassays (EIA). Time-resolved fluoroimmunoassay (TR-FIA) has been developed for several viruses.
  
  
  OBJECTIVE
  To prepare monoclonal antibodies to coronavirus strains, to incorporate them into a TR-FIA, and test the assay on clinical specimens.
  
  
  METHODS
  Monoclonal antibodies were prepared to the N nucleoprotein of the two human respiratory coronaviruses, HCV strains 229E and OC43. Monoclonals to both viruses were completely type-specific; they did not cross-react between themselves or with multiple strains of other respiratory viruses. These antibodies were configured into optimized EIA and TR-FIA tests. The all-monoclonal tests were then compared to polyclonal EIA tests in terms of their ability to detect virus in clinical specimens.
  
  
  RESULTS
  The all-monoclonal TR-FIA was uniformly the most sensitive, detecting virus in all 13 229E-positive specimens compared to 69% for the monoclonal EIA and 54% for the polyclonal EIA test. Similar results were obtained for 10 OC43-positive specimens: 100% in TR-FIA, 90% in monoclonal EIA, and 80% in polyclonal EIA. For 229E in TR-FIA, mean positive/negative (P/N) ratios were 143 for 229E-positive human embryonic lung fibroblast (HLF) cell culture fluids and 10 for positive nasopharyngeal aspirate specimens; for OC43 in TR-FIA, mean P/N values were 964 for OC43-positive rhabdomyosarcoma (RD) cell culture fluids and 174 for positive NPA specimens. The sensitivities of the TR-FIA were determined with purified virions to be 0.308 ng virus per well for HCV-229E and 0.098 ng virus per well for HCV-OC43.
  
  
  CONCLUSIONS
  This rapid and sensitive test appears to be much more sensitive than traditional antigen detection assays but will require more extensive field testing on clinical specimens.
authors.affiliation: Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, 1600(o) Clifton Rd. N.E. Atlanta, GA 30333, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Hierholzer
authors.forename: P E
authors.initials: PE
authors.lastname: Halonen
authors.forename: P G
authors.initials: PG
authors.lastname: Bingham
authors.forename: R A
authors.initials: RA
authors.lastname: Coombs
authors.forename: Y O
authors.initials: YO
authors.lastname: Stone