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Updated: Aug 8, 2026
High-throughput Detection Method for Influenza Virus
Published on: February 4, 2012
Antigen detection in human respiratory Coronavirus infections by monoclonal time-resolved fluoroimmunoassay
J C Hierholzer1, P E Halonen, P G Bingham
1Respiratory 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.
Insights
A new time-resolved fluoroimmunoassay (TR-FIA) using monoclonal antibodies offers a highly sensitive method for detecting respiratory coronaviruses. This advanced antigen detection assay shows superior performance over traditional methods in clinical specimens.
Area of Science:
- Virology
- Immunology
- Diagnostic Assays
Background:
- Viral antigen detection is crucial for diagnosing respiratory infections.
- Immunofluorescent assays (IFA) and enzyme immunoassays (EIA) are common methods.
- Time-resolved fluoroimmunoassay (TR-FIA) offers potential for improved viral detection.
Purpose of the Study:
- To develop monoclonal antibodies against human respiratory coronavirus strains (HCV 229E and OC43).
- To integrate these antibodies into a TR-FIA for enhanced sensitivity.
- To evaluate the TR-FIA's performance on clinical specimens.
Main Methods:
- Monoclonal antibodies were generated against the N nucleoprotein of HCV 229E and OC43.
- Antibodies demonstrated high type-specificity, with no cross-reactivity.
- Optimized EIA and TR-FIA tests were developed using all-monoclonal antibody configurations.
- Performance was compared against polyclonal EIA tests using clinical samples.
Main Results:
- The all-monoclonal TR-FIA achieved 100% sensitivity for both HCV 229E and OC43 in clinical specimens.
- This significantly outperformed monoclonal EIA (69-90%) and polyclonal EIA (54-80%).
- TR-FIA demonstrated high positive/negative ratios and high sensitivity with purified virions (0.308 ng/well for 229E, 0.098 ng/well for OC43).
Conclusions:
- The developed TR-FIA is a rapid and highly sensitive assay for coronavirus detection.
- It shows significantly greater sensitivity than traditional antigen detection methods.
- Further extensive field testing on clinical specimens is warranted.
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 (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.
