Abstract
The principle of time-resolved fluorometry and its application to solidphase immunoassays (TR-FIAs) was invented in the 1980s in Turku, Finland at the Wallac Biochemical Laboratory and University of Turku (1,2). One of the early diagnostic applications was the detection of viral antigens with immunoreagents prepared from either polyclonal or monoclonal antibodies (3–7). Viral antigen detection using time-resolved fluorescence combined with one-step immunoassay has resulted in the highest sensitivities obtained with any solid-phase immunoassays. In the test, Eu-labeled monoclonal antibody reacts quickly and efficiently in liquid-phase with the viral antigen of the test specimen and the formed antigen-antibody complex is adsorbed to another monoclonal antibody attached to solid-phase (Fig. 1). The bound Eu-chelate is then measured with a single-photon-counting fluorometer designed to measure only the specific lanthanide fluorescence with a long decay time. This is achieved when the background fluorescence reaches an insignificant level, The specificity of fluorescence is further increased by the large difference between the excitation and emission wavelengths of lanthanide compounds (Stoke’s shift).
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Soini, E and Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates-A new generation of nonisotopic immunoassays. Clin Chem 29, 65–68
Soini, E. and Lovgren, T. (1987) Time-resolved fluorescence of lanthanide probes and applications in biotechnology CRC Crit Rev Anal Chem. 18, 105–154.
Siitari, H., Hemmila, I, Lovgren, T., and Koistinen, V (1983) Detection of hepatitis-B surface antigen using time-resolved fluoroimmunoassay. Nature 301, 258–260
Halonen, P., Meurman, O, Lovgren, T., Hemmila, I, and Soini, E. (1983) Detection of viral antigens by time-resolved fluoroimmunoassay Cur Top. Microbiol Immunol 104, 133–146
Halonen, P, Obert, G, and Hierholzer, J. C. (1985) Direct detection of viral antigens by immunoassays: a four-year experience and new developments, in Medical virology IV (de la Maza, L M. and Peterson, E. M, eds.), Lawrence Erlbaum, Hillsdale, NJ, pp. 65–83.
Walls, H. K, Johansson, K. H, Harmon, M. W, Halonen P. E., and Kendal, A. P. (1986) Time-resolved fluoroimmunoassay with monoclonal antibodies for rapid diagnosis of influenza infections. J Clin Microbiol 24, 907–912.
Hierholzer, J C, Johansson, K H, Anderson, L J, Tshou, C. J, and Halonen, P. E. (1987) Comparison of monoclonal time-resolved fluoroimmunoassay with monoclonal capture-biotinylated detector enzyme immunoassay for adenovirus antigen detection. J. Clin Microbiol. 25, 1662–1667
Dahlen, P. O, Iitra, A J, Skagius, G., Frostell, Å, Nunn, M. F, and Kwiatkowski, M (1991) Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assay. J Clin Microbiol 29, 798–804.
Hurskainen, P., Dahlen, P, Ylikoski, J., Kwiatkowski, M., Sutari, H., and Lovgren, T. (1991) Preparation of europium-labelled DNA probes and their properties Nucleic Acids Res 19, 1057–1061.
Halonen, P., Rocha, E., Hierholzer, J., Holloway, B., Hyypiä, T., Hurskainen, P., and Pallansch, M. (1995) Detection of enteroviruses and rhinoviruses in clinical specimens by PCR and liquid-phase hybridization. J, Clin Microbiol 33, 648–653
Lovgren, T., Iitiä, A., Hurskainen, P., and Dahlén, P. (1995) Detection of lanthanide chelates by time-resolved fluorescence, in Nonisotopic Probingg, Blotting and Sequencing, 2nd ed. (Kricka, L., ed.), Academic, San Diego, pp. 331–376.
Sambrook, J., Fritsch, E. F, and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual, 2nd ed. (Nolan, C., Ford, N., and Ferguson, M., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Agrawal, S., Christodoulou, C., and Gait, M. J. (1986) Efficient methods for attaching nonradioactive labels to the 5′ ends of synthetic oligodeoxyribonucleotides Nucleic Acids Res 14, 6227–6245
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© 1998 Humana Press Inc.
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Halonen, P., Lövgren, T. (1998). Time-Resolved Fluorescence. In: Stephenson, J.R., Warnes, A. (eds) Diagnostic Virology Protocols. Methods in Molecular Medicine™, vol 12. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-479-8:245
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DOI: https://doi.org/10.1385/0-89603-479-8:245
Publisher Name: Springer, Totowa, NJ
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