ELSEVIER Clinical and Diagnostic Virology 2 (1994) 165-179 Clinical and Diagnostic Virology Antigen detection in human respiratory Coronavirus infections by monoclonal time-resolved fluoroimmunoassay John C. Hierholzer *, Pekka E. Halonen a,b, Patricia G. Bingham , Richard A. Coombs , Yvonne O. Stone Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, 1600Clifton Rd. N.E., Atlanta, GA 30333, USA; b Department of Virology, University of Turku, Kiinamyllynkatu 13, SF-20520, Turku 52, Finland Received 27 May 1993; revised 18 November 1993; accepted 1 December 1993 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. Objectives: To prepare monoclonal antibodies to coronavirus strains, to incorporate them into a TR-FIA, and test the assay on clinical specimens. Study design: 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 (PfN) 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. *Corresponding author. 0928-0197/94/$7.00 1994 Elsevier Science B.V. All rights reserved SSDI 0928-0197 (94)E0050-P 166 Ji (2 Hierholzer et al./Clinical and Diagnostic Virology 2 (1994) 165-179 Key words. Human coronavirus; Monoclonal antibody; Rapid antigen test; Immunoassay 1. Introduction Rapid testing for the respiratory viruses has become widely appreciated in the clinical laboratory as virus-specific indirect fluorescent antibody (IFA) and spot enzyme immunoassay (EIA) kits have become commercially available and quantita- tive tests such as EIA and time-resolved fluoroimmunoassay (TR-FIA) have been improved. Both of these tests are of proven greater sensitivity than IFA, and TR-FIA performed with monoclonal antibodies has been shown to be of greater sensitivity even than similarly constructed EIA tests (reviewed in Hierholzer et al., 1990; Mclntosh et al., 1993). We and others have described monoclonal TR-FIA tests for influenza, adenovirus, respiratory syncytial virus, parainfluenza l-4, mumpsvirus, and the enteroviruses responsible for acute hemorrhagic conjunctivitis (Halonen et al., 1985, 1989; Walls et al., 1986; Hierholzer et al., 1987, 1989, 1990, 1993; Brown et al., 1990; Siitari, 1990; Bucher et al., 1991). Coronaviruses are another group of viruses that cause widespread respiratory infections and thus should be included in the TR-FIA battery for rapid viral diagnosis. Of the four recognized human coronavirus strains, only two have been characterized, and both are associated with upper and occasionally lower respiratory tract disease. IFA and EIA tests for these strains, called human coronavirus (HCV) 229E and HCV-OC43, have been described and applied to epidemiologic surveys with good success (McIntosh et al., 1978; Isaacs et al., 1983; Macnaughton et al., 1983). In the present report, we describe the production of monoclonal antibodies to HCV-229E and HCV-OC43 and describe their use in IFA, EIA, and TR-FIA, with emphasis on the TR-FIA as a highly-sensitive rapid anti- gen test. 2. Materials and methods Viruses and cell culture The prototype strain of HCV-229E was initially obtained from Dorothy Hamre, University of Chicago, at the sHKzWI3811 passage level (Hamre and Procknow, 1966). It has been maintained in a variety of human embryonic lung diploid fibroblast cell lines, e.g., WI38, RU-1, MRC-5, HELF, HLF. The present studies were