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Time-resolved fluoroimmunoassay

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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.

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References

  1. Soini, E. and Hemmilä, I. (1979) Fluoroimmunoassay: present status and key problems. Clin. Chem. 25, 353–362.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  3. Ekins, R. P. and Dakubu, S. (1985) The development of high sensitivity pulsed light, time-resolved fluoroimmunoassays. Pure Appl. Chem. 57, 473–482.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  9. Diamandis, E. P. (1988) Immunoassays with time-resolved fluorescence spectroscopy: principles and applications. Clin. Biochem. 21, 139–150.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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. Harris, M. I. and Coleman, J.E. (1968) The biosynthesis of Apo-and metalloalkaline phosphatase of Escherichia coli. J. Biol. Chem. 243, 5063–5073.

    PubMed  CAS  Google Scholar 

  14. Krejcarek, G. E. and Tucker, K. L. (1977) Covalent attachment of chelating group to macromolecules. Biochem. Biophys. Res. Commun. 77, 581–585.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. National Institute for Research on Cancer, 10 Viale Benedetto XV, 16132, Genoa, Italy

    Paolo Degan

  2. Department of Anatomy, Biochemistry and veterinary Physiology, University of Pisa, 2 Viale delle Piagge, 56124, Pisa, Italy

    Adriano Podestà & Giorgio Montagnoli

Authors
  1. Paolo Degan
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  2. Adriano Podestà
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  3. Giorgio Montagnoli
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Correspondence to Giorgio Montagnoli.

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Degan, P., Podestà, A. & Montagnoli, G. Time-resolved fluoroimmunoassay. Mol Biotechnol 13, 215–222 (1999). https://doi.org/10.1385/MB:13:3:215

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  • DOI: https://doi.org/10.1385/MB:13:3:215

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