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Journal of Clinical Microbiology, November 2007, p. 3581-3588, Vol. 45, No. 11
0095-1137/07/$08.00+0     doi:10.1128/JCM.00128-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid Method for Detection of Influenza A and B Virus Antigens by Use of a Two-Photon Excitation Assay Technique and Dry-Chemistry Reagents{triangledown}

Janne O. Koskinen,1,2,3 Raija Vainionpää,3 Niko J. Meltola,2 Jori Soukka,4 Pekka E. Hänninen,2 and Aleksi E. Soini1,3*

Turku University of Applied Sciences, Life Sciences, Turku, Finland,1 Laboratory of Biophysics, Institute of Biomedicine, and Medicity Research Laboratories, University of Turku, Turku, Finland,2 Department of Virology, University of Turku, Turku, Finland,3 Arctic Diagnostics Oy, Turku, Finland4

Received 18 January 2007/ Returned for modification 18 March 2007/ Accepted 31 August 2007

New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.

* Corresponding author. Mailing address: ABAI-Laboratory, Turku University of Applied Sciences, Lemminkäisenkatu 30, 20520 Turku, Finland. Phone: 358 40 5510424. Fax: 358 22 6371250. E-mail: aleksi.soini{at}arcdia.com

{triangledown} Published ahead of print on 12 September 2007.

Journal of Clinical Microbiology, November 2007, p. 3581-3588, Vol. 45, No. 11
0095-1137/07/$08.00+0     doi:10.1128/JCM.00128-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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