Organic compounds -- part of the class 532-570 series – Organic compounds – Rare earth containing
Reexamination Certificate
2000-01-18
2002-01-22
Hartley, Michael G. (Department: 1619)
Organic compounds -- part of the class 532-570 series
Organic compounds
Rare earth containing
C424S009600, C424S009610, C436S172000, C600S317000
Reexamination Certificate
active
06340744
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the invention
This invention relates to enhanced luminescence of covalently coupled dyes containing lanthanide macrocycle complexes, especially as tags for detecting members of combining pairs, and to the detection of low levels of these dyes.
2. Prior Art
The sensitivity of fluorescence measurements for the analysis of biological samples is often limited by background signal due to auto fluorescence or Raman scattering.
At present, the microscopic visualization of luminescent labels containing lanthanide (III) ions, primarily europium (III), as light emitting centers is best performed with time-gated instrumentation, which by virtually eliminating the background fluorescence, results in an improved signal to noise ratio. Although the use of time-gated luminescence for microscopic and clinical chemistry analyses holds the promise of maximizing the detectability and quantitation of markers containing lanthanide complexes, this instrumentation is costly and not widely available; furthermore, time-gated measurements often involve loss of signal or precision. The combination of an image-intensifier coupled to a CCD permits the high speed gating of image acquisition. Briefly, rapid voltage changes at the intensifier dynodes result in concomitant changes in the current amplification, which effectively shutters the photoelectrons impinging on the phosphor that is located directly in front of the CCD surface. Other time-gating approaches to control the image acquisition of digital microscopes include high speed rotating choppers and ferro-electric shutters. However, none of these is suitable for the clinical pathology or clinical chemistry laboratory.
Vallarino and Leif U.S. Pat. Nos. 5,373,093 and 5,696,240 disclosed hexa-aza-macrocyclic complexes incorporating a lanthanide, actinide, or yttrium ion possessing high kinetic stability and pendant functional groups that can be readily coupled/conjugated to a biologically active molecule such as an antibody or antigen, or to a biologically compatible ionically uncharged macromolecule such as a linear or cross-linked polysaccharide. The complexes of europium (III) and terbium (III), for example, possess a long-lived fluorescence intensity that can be substantially increased by interaction with a suitable enhancer, for example the sodium salt of dibenzoylmethane or thenoyltrifluoroacetyl-acetone. The entire disclosure of these patents is here incorporated by reference.
Xu U.S. Pat. No. 5,316,909 disclosed the interaction between B-diketonate complexes of luminescent lanthanide (III) and yttrium (III) in the presence of a synergistic compound to provide a cofluorescence effect which significantly increases the emission intensity. The embodiment described requires that the original chelate containing the fluorescent ion be dissociated from the biomolecule, followed by the addition of the yttrium cofluorescence species and incubation with the synergistic compounds for 1 to 15 minutes prior to measurement.
Adeyiga et al. SPIE vol. 2678, pages 212-220 (1996) disclosed extension of Xu's method to other lanthanide complexes including the europium (III) macrocycles. It is stated at page 215 that “a preliminary, unoptimized study with the parent unfunctionalized macrocycle {Eu(C
22
H
26
N
6
)} triacetate complex as prototype has shown an approximate three fold increase in luminescence.”
SUMMARY OF THE INVENTION
In accordance with this invention, there is provided a spectrofluorimetrically detectable luminescent composition comprising water, a micelle-producing amount of at least one surfactant, at least 1×10
10
moles/liter of at least one energy transfer acceptor lanthanide element macrocycle compound having an emission spectrum peak in the range from 500 to 950 nanometers, and a luminescence-enhancing amount of at least one energy transfer donor compound of yttrium or a 3-valent lanthanide element having atomic number 59-71, provided that the lanthanide element of said macrocycle compound and the lanthanide element of said energy transfer donor compound are not identical.
The enhanced luminescence of compositions according to the invention pennits the detection and/or quantitation of the lanthanide macrocycle compound and complexes thereof without the use of expensive and complicated time-gated detection systems. As a result, these macrocycle compounds and complexes thereof are useful as reporter molecules in immunoassays, analytical cytology, histological staining, and imaging processing.
The enhanced luminescence of compositions according to the invention caused by a different lanthanide energy transfer donor compound can also occur with functionalized derivatives of energy transfer acceptor lanthanide macrocycles, that is macrocycles substituted with reactive functional groups at which reaction with analytes can take place; with reaction products of such functionalized macrocycles with such analytes; and with polymers which contain multiple lanthanide-containing units. Through their reactive functional groups, functionalized energy transfer acceptor lanthanide macrocycles can be attached by a coupling functionality to analytes including small molecules of biological interest having molecular weights from 125 to 2000 daltons, such as nucleic acid bases or haptens, and large molecules of biological interest having molecular weights greater than 2000 daltons such as proteins including antibodies, polysaccharides, or nucleic acids.
Also in accordance with this invention, there is provided a method for analysis of a sample suspected of containing at least one analyte, frequently a biologically active compound, said method comprising:
a) contacting said sample with a functionalized complex of a metal M, where M is a metal ion selected from the group consisting of a lanthanide having atomic number 57-71, an actinide having atomic number 89-103 and yttrium(III) having atomic number 39;
in a reaction medium under binding conditions, whereby said analyte when present either interacts with said complex to form a conjugate or competes for interaction with a binding material specific for interaction with said complex and with said analyte;
b) adding to said reaction medium a luminescence-enhancing amount of at least one energy transfer donor compound of yttrium or a 3-valent lanthanide element having atomic number 59-71, provided that the lanthanide element of said macrocycle compound and a lanthanide element of said energy transfer donor compound are not identical,
(c) subjecting said reaction medium to excitation energy in the range of 200-400 nm, whereby enhanced luminescence in the range of 500-950 nm is generated,
(d) monitoring said luminescence of the reaction medium to measure in said sample at least one of the following:
(1) presence and/or concentration of said conjugate;
(2) presence and/or concentration of the product of the interaction of said complex with said binding material; and
(3) presence and/or concentration of the product of the interaction of the conjugate with the binding material.
It is a feature of this invention that the method does not require prior dissociation of the luminescence-enhanced complex before measuring its emission spectrum. Moreover, since the excitation spectra of lanthanide macrocycles and those of several DNA-specific dyes, including 4′, 6-diamidino-2-phenylindole (DAPI) occur in the same region of the ultraviolet, both types of compounds can be excited at the same wavelength, while their emission spectra are very different. The organic dyes have broad emissions in the blue region of the spectrum while the enhanced luminescence of lanthanide macrocycles according to this invention occurs as very narrow emission peaks in the red. This difference allows the major emission of the enhanced luminescence composition of this invention to be unambiguously detected even when its intensity is much lower than that of the very strong emission of the DNA specific organic dyes.
It is a further feature of the invention that the composition and method of the in
Leif Robert C.
Vallarino Lidia
Hartley Michael G.
Hibnick Gerald R.
Kauder Otto S.
Schwartz Robert M.
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