Fluorescence Energy Transfer Immunoassays

  • Pyare L. Khanna


Homogeneous immunoassays have gained broad acceptance during the past decade and are being widely utilized for both quantitative and qualitative analysis of a wide variety of analytes in the clinical laboratory. Concurrent to the development of enzyme immunoassays, homogeneous fluorescence immunoassays have played an equally important role in the development of a wide variety of immunochemical methods. One of the earliest procedures of histochemical staining using fluorescent labels (Coones, Creech and Jones, 1941) is still a method of choice for immunochemical localization of tissue antigens. After incubation of tissue antigens with fluorescer-labeled primary or secondary antibody, the excess of the labeled reagent is washed away and the resulting stained immune complexes can be visualized under a dark field microscope. This method, although cumbersome as an immunoassay technique, does provide useful qualitative data. Such use of receptor groups in competitive immunoassays was not further exploited until 1959 when the technique of radioimmunoassays was introduced (Berson and Yallow, 1959). The basic technique involves competition of the analyte with a radio-labeled antigen for a limited number of binding sites. After separation of free and bound antigen and measurement of the radioactivity of these fractions, the quantitation can be achieved by comparison with measurements obtained from a set of known standards. This method provided the ability to determine antigens in solutions quantitatively. Although various modifications have been developed during the past years, the basic technique suffers from the short shelf life of the radioisotopes and the complex labor intensive protocols involving separation of the bound and free fractions prior to measurement. More recently, there has been considerable interest in the use of nonisotopic labels for immunoassays. These have included enzymes (Scharpe, Cooneman and Bloome, 1976; Wisdom, 1976; Van Weeman and Schuurs, 1971), metals (Cais, Dari, Eder, et al, 1977), chemiluminescent groups (Simpson, Campbell and Ryall, 1979), and fluorescent groups (Soini and Hemmila, 1979).


High Quantum Yield Efficient Energy Transfer High Extinction Coefficient Total Fluorescence Intensity Label Antigen 
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  1. Berson, S. A. and Yalow, R. S., 1959, Quantitative aspects of the reaction between insulin and insulin binding antibodies, J. Clin. Invest. 38, 1196–2016.Google Scholar
  2. Cais, N., Dari, S. and Eder, Y., et al, 1977, Metalloimmunoassay, Nature 270, 543–635.CrossRefGoogle Scholar
  3. Coons, A. H., Creech, H. J. and Jones, R. N., 1941, Immunological properties of an antibody containing a fluorescent group, Proc. Soc. Exp. Biol. Med. 47, 200–202.Google Scholar
  4. Dandliker, W. B. and Geiger, G., 1961, Quantification of the antigen-antibody reaction to the polarization of fluorescence, Biochem. Biophys. Res. Commun. 5, 299–304PubMedCrossRefGoogle Scholar
  5. Forster, T., 1948, Zwischenmolekulare energiewanderung und fluoreszenz, Ann. Phys. (Leipzig), 2, 55–75.Google Scholar
  6. Gantt, E., 1981, Phycobilisomes, Ann. Rev. Plant. Physiol. 32, 327–347.CrossRefGoogle Scholar
  7. Hendrix, J. L., 1983, Porphyrins and chlorophylls as probes for fluoroimmunoassays, Clin. Chem. 29, 1003.PubMedGoogle Scholar
  8. Molecular Probes Catalogue, Junction City, Oregon 97448.Google Scholar
  9. Oi, V. T., Glazer, A. N. and Stryer, L., 1982, Fluorescent phycobiliprotein conjugates for analytes of cells and molecules, J. Cell Biology 93, 981–986.CrossRefGoogle Scholar
  10. Rodgers, R., Schwarzberg, M., Khanna, P. L., Chang, C. H. and Ullman, E., 1978, A fluorescence quenching immunoassay for human IgA, Clin. Chem. 24, 1033.Google Scholar
  11. Scharpe, S. L., Cooneman, W. M. and Bloome, W. J., 1976, Quantitative enzyme immunoassays: current status, Clin. Chem. 22, 733–738.PubMedGoogle Scholar
  12. Simpson, J. S. A., Campbell, A. K. and Ryall, M. E. T., 1979, A stable chemiluminiscent labelled antibody for immunological assays, Nature 279, 646–647.PubMedCrossRefGoogle Scholar
  13. Soini, E. and Hemmila, I., 1979, Fluoroimmunoassay: present status and key problems, Clin. Chem. 25, 353–361.PubMedGoogle Scholar
  14. Ullman, E. F. and Khanna, P. L., 1981, Fluorescence excitation transfer immunoassay, Methods in Enzymology 74, 28–60.PubMedCrossRefGoogle Scholar
  15. Ullman, E. F., Schwarzberg, M. and Rubenstein, K. E., 1976, Fluorescence excitation transfer immunoassay, J. Biol. Chem. 251, 4172–4178.PubMedGoogle Scholar
  16. Van Weeman, B. K., and Schuurs, A. H., 1971, Immunoassay using antigenenzyme conjugates, FEBS. Lett. 15, 232–236.CrossRefGoogle Scholar
  17. Weider, I., 1978, Immunofluorescence and related staining techniques, Elsevier/North Holland Biomedical Press, 67.Google Scholar
  18. Wisdom, G. B., 1976, Enzyme immunoassay, Clin. Chem. 22, 1243–1255.PubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1988

Authors and Affiliations

  • Pyare L. Khanna
    • 1
  1. 1.SyvaPalo AltoUSA

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