Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-12-21
1998-12-29
Ceperley, Mary E.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 75, 436500, G01N 33535, G01N 3378, G01N 33545
Patent
active
058540083
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to fluorescent lanthanide metal chelates and their use in diagnostics.
BACKGROUND OF THE INVENTION
In order to facilitate discussion of known fluorescent entities, their application and the manner in which the fluorescent entities in this invention distinguish from known systems, reference will be made to several scientific articles throughout the specification. To facilitate such reference the articles have been listed by number as follows: 57A. Biochem. 137, 335-343. C.; Diamandis, E. P. Clin. Biochem. 1988, 21, 173-178. 1987, 33, 2281-2283. Chem. 1989, 61, 48-53. 1991, 197, 213-224. Clin. Chem. 1992 (in press). Biochem. 1989, 26, 238-243.
Fluorescent lanthanide metal chelates and in particular those for europium and terbium chelates have significant commercial application because of their use as labels in highly sensitive time-resolved fluorometric immunoassays (1-4). The first commercially available time-resolved fluorescence immunoassay system, Delfiae.RTM. (available by Pharmacia-LKB, Sweden) uses Eu.sup.3+ as immunological label (5,6). A second-generation time-resolved fluorometric immunoassay system, FlAgene.RTM. (available by CyberFluor Inc., Toronto, Canada, the applicant in this application) uses the europium chelator 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as immunological label (7.8). These two systems, along with the principles of time-resolved fluorometry and its application to immunoassay and other bioanalytical techniques have been investigated in detail (1-4).
Recently, Tb.sup.3+ and its chelates have been used as immunological and nucleic acid labels (9,10). In general, Tb.sup.3+ is inferior to Eu.sup.3+ in terms of detectability. However, Eu.sup.3+ and Tb.sup.3+ can be used simultaneously for dual analyte assays (11).
The mechanism of fluorescence of the Eu.sup.3+ and Tb.sup.3+ chelates has been described (1-4). These two ions, when excited by radiation, emit very weak metal ion fluorescence which is not analytically useful. The fluorescence is dramatically enhanced when the metal ion forms a chelate with appropriate organic ligands. An important property of these chelates is that the radiation is absorbed at a wavelength characteristic of the ligand and is emitted as a line spectrum characteristic of the metal ion. This is due to an intramolecular energy transfer from the ligand to the central metal ion (12). In general, it is difficult to predict theoretically which organic molecules-ligands can form highly fluorescent complexes with lanthanide metals, and in particular, with Eu.sup.3+ and Tb.sup.3+. Some classes of compounds i.e. the diketones, tetracyclines, phenanthrolines, acetylene derivatives, five-membered heterocyclic ring derivatives, benzoic acid derivatives, biphenyl derivatives, pyridine derivatives, pyrimidine and pyrazine derivatives, di-and tripyridyl derivatives, quinoline derivatives, aza-uracil and purine derivatives and phosphorimido-derivatives have been identified as fluorogenic ligands of Eu.sup.3+ and/or Tb.sup.3+ (13).
In time-resolved fluorometric immunoassays, it is desirable to use a chelate-label which can be detected down to the subpicomolar range (5). Alternatively, multiple labelling strategies can be used in order to achieve subpicomolar analyte sensitivity (1 and 4-16). More recently, there have been efforts to combine time-resolved fluorometric immunoassay with enzymatic catalysis. In one version of this approach, the primary immunological label is alkaline phosphatase (ALP); its substrate is the phosphate ester of 5-fluorosalicylic acid (FSAP). The nonhydrolysed ester (FSAP) and the hydrolysed ester (FSA) have different behaviour in Tb.sup.3+ -EDTA solutions. When FSA is added to an aqueous alkaline solution containing Tb.sup.3+ -EDTA, a mixed complex is formed which emits long-lived fluorescence characteristic of Tb.sup.3+. FSA is an appropriate ligand for energy transfer to the metal ion. An intact hydroxyl group on the FSA molecule is essential for these highly
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Ceperley Mary E.
Nordion International Inc.
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