Molecular Biotechnology

, Volume 13, Issue 3, pp 215–222 | Cite as

Time-resolved fluoroimmunoassay

  • Paolo Degan
  • Adriano Podestà
  • Giorgio Montagnoli
Protocol

Abstract

A method is reported to set up a standard competitive TR-FIA. A simple and inexpensive way to prepare reagents and carry out operations is presented as well, with the aim to make it possible to perform a very sensitive analytical procedure in a personalized way within a nondedicated biochemistry laboratory. This protocol is general and can be easily modified with consideration to the analytical target. Once the antibody is available, both its labeling with diethylenetriaminepentaacetic acid dianhydride and Eu3+, and the setting-up of the assay with measurement of europium ion time-resolved fluorescence in a home-made enhancement solution become feasible.

Index Entries

Fluoroimmunoassay, time-resolution europium ion flourescence enhancement 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Soini, E. and Hemmilä, I. (1979) Fluoroimmunoassay: present status and key problems. Clin. Chem. 25, 353–362.PubMedGoogle Scholar
  2. 2.
    Hemmilä, I., Dakubu, S., Mukkala, V.-M., Siitari, H., and Lövgren, T. (1984) Europium as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137, 335–343.PubMedCrossRefGoogle Scholar
  3. 3.
    Ekins, R. P. and Dakubu, S. (1985) The development of high sensitivity pulsed light, time-resolved fluoroimmunoassays. Pure Appl. Chem. 57, 473–482.CrossRefGoogle Scholar
  4. 4.
    Mukkala, V.-M., Mikola, H., and Hemmilä, I. (1989) The synthesis and use of activated N-benzyl derivatives of diethylenetriaminetetraacetic acids: alternative reagents for labeling of antibodies with metal ions. Anal. Biochem. 178, 319–325.CrossRefGoogle Scholar
  5. 5.
    Keelan, J. A., France, J. T., and Barling, P. M. (1987) An alternative fluorescence enhancement solution for use in lanthanide-based time resolved fluoroimmunoassays. Clin. Chem. 33, 2292–2295.PubMedGoogle Scholar
  6. 6.
    Degan, P., Abbondandolo, A., and Montagnoli, G. (1990) A new fluorescence enhancement solution for europium-based time-resolved fluoroimmunoassays J. Biolum. Chemilum. 5, 207–212.CrossRefGoogle Scholar
  7. 7.
    Evangelista, R. A., Pollak, A., Brian, A., Templeton, E. F., Morton, R. C., and Diamandis, E. P. (1988) A new europium chelate for protein labelling and time-resolved fluorometric applications. Clin. Biochem. 21, 173–178.PubMedCrossRefGoogle Scholar
  8. 8.
    Diamandis, E. P., Morton, R. C., Reichstein, E. and Khosravi, M. J. (1988) Multiple fluorescence labeling with europium chelators. Application to time-resolved fluoroimmunoassays. Anal. Chem. 61, 48–52.CrossRefGoogle Scholar
  9. 9.
    Diamandis, E. P. (1988) Immunoassays with time-resolved fluorescence spectroscopy: principles and applications. Clin. Biochem. 21, 139–150.PubMedGoogle Scholar
  10. 10.
    Ferguson, R. A., Yu He, Kalyvas M., Zammit S., and Diamandis E. (1996) Ultra sensitive detection of prostate-specific antigen by a time-resolved immunofluorometric assay and the Immulite immunochemiluminescent third-generation assay: potential applications in prostate and breast cancers. Clin. Chem. 42, 675–684.PubMedGoogle Scholar
  11. 11.
    Degan, P., Montesano, R., Abbondandolo, A., and Montagnoli, G. (1990). Time resolved fluoroimmunoassay of 7-methyl-2′-deoxyguanosine imidazole (ring open) J. Immunol. Methods 133, 119–125.PubMedCrossRefGoogle Scholar
  12. 12.
    Ekins, R. (1981) Merits and disadvantage of different labels and methods of immunoassay, in Immunoassays for the ’80s (Voller, A. Bartlett, A., and Bidwell, D., eds.), MTP Press, Lancaster, pp. 5–16.Google Scholar
  13. 13.
    Harris, M. I. and Coleman, J.E. (1968) The biosynthesis of Apo-and metalloalkaline phosphatase of Escherichia coli. J. Biol. Chem. 243, 5063–5073.PubMedGoogle Scholar
  14. 14.
    Krejcarek, G. E. and Tucker, K. L. (1977) Covalent attachment of chelating group to macromolecules. Biochem. Biophys. Res. Commun. 77, 581–585.PubMedCrossRefGoogle Scholar
  15. 15.
    Ius, A., Ferrara, L., Meroni, G., and Bacigalupo, M. (1985) Eu-conjugated protein-A as a label of IGg antibodies Fresenius Z. Anal. Chem. 322, 509–510.CrossRefGoogle Scholar
  16. 16.
    Hemmilä, I., Holttinen, S., Pettersson, K., and Lövgren, T. (1987) Double-label time-resolved immunofluorometry of lutropin and follitropin in serum. Clin. Chem. 33, 2281–2283.PubMedGoogle Scholar
  17. 17.
    Bador, R., Déchaud, H., Claustrat, F., and Desuzinges, C. (1987) Europium and samarium as labels in time-resolved immunofluorometric assay of follitropin. Clin. Chem. 33, 48–51.PubMedGoogle Scholar

Copyright information

© Humana Press Inc 1999

Authors and Affiliations

  • Paolo Degan
    • 1
  • Adriano Podestà
    • 2
  • Giorgio Montagnoli
    • 2
  1. 1.National Institute for Research on CancerGenoaItaly
  2. 2.Department of Anatomy, Biochemistry and veterinary PhysiologyUniversity of PisaPisaItaly

Personalised recommendations