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WO1998039654A2 - Complexes de ligands-ions de neodymium (iii), ytterbium (iii) ou erbium (iii) - Google Patents

Complexes de ligands-ions de neodymium (iii), ytterbium (iii) ou erbium (iii)

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Publication number
WO1998039654A2
WO1998039654A2 PCT/EP1998/001287 EP9801287W WO9839654A2 WO 1998039654 A2 WO1998039654 A2 WO 1998039654A2 EP 9801287 W EP9801287 W EP 9801287W WO 9839654 A2 WO9839654 A2 WO 9839654A2
Authority
WO
WIPO (PCT)
Prior art keywords
derivatives
ion
lll
analyte
lanthanide
Prior art date
Application number
PCT/EP1998/001287
Other languages
English (en)
Other versions
WO1998039654A3 (fr
Inventor
Johannes Willem Hofstraat
Original Assignee
Akzo Nobel N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Akzo Nobel N.V. filed Critical Akzo Nobel N.V.
Priority to AU68284/98A priority Critical patent/AU6828498A/en
Priority to EP98913667A priority patent/EP0968424A1/fr
Publication of WO1998039654A2 publication Critical patent/WO1998039654A2/fr
Publication of WO1998039654A3 publication Critical patent/WO1998039654A3/fr
Priority to US09/380,336 priority patent/US6951760B2/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/40—Rare earth chelates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/969—Multiple layering of reactants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/971—Capture of complex after antigen-antibody reaction

Definitions

  • the invention pertains to lanthanide ion-ligand complexes, in particular to neodymium(lll) ion (Nd 3+ ), ytterbium(lll) ion (Yb 3+ ), or erbium(lll) ion (Er 3+ ) ligand complexes, to the use of said lanthanide ion-ligand complexes for the manufacture of a diagnostic kit, to a diagnostic kit comprising the same, and to a method of detecting an analyte in a matrix of biomedical interest.
  • the other members of the lanthanide metals are considered to be unsuitable as luminescent probes because they have much smaller gaps between the excited states and the ground state.
  • the lanthanides are coordinated to form complexes with ligands, which complexes often are unstable in aqueous solutions.
  • the excitation energy used regularly interferes with the biological material which is used for diagnostic purposes, which more often than not is sensitive to light of the UV region. Further it is of advantage to have a metal ion which exhibits a long lifetime in the excited state, which requires minimization of quenching and back- transfer.
  • lanthanide ion-ligand complex comprises neodymium(lll) ion (Nd 3+ ), ytterbium(lll) ion (Yb 3+ ), or erbium(lll) ion (Er 3+ ) as the lanthanide ion, and the ligand comprises or is in contact with a sensitizing moiety which absorbs in the 400-1000 nm region, and preferably in the 400-800 nm region
  • the complex displays long-lived near-IR luminescence without having the disadvantages of the prior art methods.
  • These lanthanide ion-ligand complexes further comprise an (immuno)- reactant for attachment to an analyte.
  • the ligand When the ligand is in contact with the sensitizing moiety, it may be covalently or ionogenically bonded, or the ligand may be in such close vicinity to the sensitizing moiety that energy transfer between the lanthanide ion-ligand complex and the sensitizing moiety is possible.
  • These lanthanide ion-complexes can make use of inexpensive 400-1000 nm lasers or other light sources, emit luminescence in the near-IR spectrum, have long luminescence lifetimes, high sensitivity, and good stability with respect to the irradiated light and towards the solvents used, especially towards aqueous solutions.
  • the lanthanide ion-complexes according to this invention have long luminescence lifetimes and do emit in the near-IR spectrum, which leads to "zero-interference" detection (necessary for diagnostic purposes). Further, the lanthanide ion-complexes according to this invention very efficiently quench the excited triplet state of the sensitizer, which is a major source of singlet oxygen leading to photo-oxidative damage, and therefore enhance stability.
  • luminescence is light which is emitted by a compound upon excitation by any means, among them irradiation of laser light.
  • Luminescence lifetime is the time in which the luminescence emission intensity has decayed to 1/e of its original value.
  • Luminescence quenching is the process which leads to radiationless deactivation of the luminescent excited state, for instance, as a result of collisions of the excited molecules with species which accept energy from the excited state and dispose of it non-radiatively.
  • the ligand comprises a complexing moiety which shields the ion from quenching by solvent molecules, in particular water which is generally present in bioassays and contains the OH moiety with particular quenching ability, and provides strong binding of the ion and an anchor for attachment to the (immuno)reactant.
  • the attachment of the (immuno)reactant to the lanthanide ion-ligand complex can be performed by the conventional methods well-known in the art.
  • the ligand further comprises a sensitizing moiety, which is efficiently excited and able to transfer energy to the lanthanide.
  • the sensitizing moiety should be as close to the ion as possible to make the energy transfer process more efficient.
  • the ligand may be a complex forming moiety which is in contact with, but not bonded to the sensitizer.
  • the sensitizer comprises a site which can act as the complexing moiety.
  • the sensitizing moiety may be any sensitizing moiety that absorbs light in the visible or near-IR range of the spectrum, i.e. between about 400 and 1000 nm, and more preferably between about 400 and 800 nm.
  • the sensitizing moiety is selected from fluorescein derivatives such as fluorexon, eosin, erythrosin, fluorescein, rose bengal, calcium green, and oregon green; triphenylmethane derivatives such as methylthymol blue, xylenol orange, brilliant blue, methyl green, and malachite green; porphyrin derivatives; rhodamine derivatives such as rhodamine 6G, tetrabromo- rhodamine, and lissamine; phenothiazine derivatives such as thionin and methylene blue; phenoxazine derivatives such as nile blue; coumarin derivatives; acridin derivatives such as acridin orange; (thio)indigo derivatives; carbocyanine derivatives; squaraine derivatives; and (na)phthalocyanine derivatives.
  • fluorescein derivatives such as fluorexon, eos
  • Coumarin derivatives include 2- and 4-coumarins such as coumarin 120, 124, 445, 450, 490, 500, 503, and trifluoromethylcoumarin.
  • Other sensitizers which absorb in the visible region ca ⁇ also be employed.
  • the ligand may be a composition comprising any compound comprising oxygen, nitrogen, phosphorous, or sulfur moieties which have complexing ability towards Nd(lll), Yb(lll), or Er(lll) ions, in particular polyaminocarboxylic acid, pyridinedicarboxylic acid (dipicolinic acid, DPA), or a derivative thereof, and a sensitizer selected from fluorescein derivatives such as fluorexon, eosin, erythrosin, fluorescein, rose bengal, calcium green, and Oregon green; triphenylmethane derivatives such as methylthymol blue, xylenol orange, brilliant blue, methyl green, and malachite green; porphyrin derivatives; rhodamine derivatives such as rhodamine 6G, rhodamine B, tetrabromo- rhodamine, and Iissamine; phenothiazine derivatives such as thionin
  • Suitable polyaminocarboxylic acids are, for instance, ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DPTA), and triethylenetetraamine hexaacetic acid (TTHA), all basically comprising the aminocarboxylate groups as complexing moieties. These compounds have excellent solubility and stability in water.
  • EDTA ethylenediamine tetraacetic acid
  • DPTA diethylenetriamine pentaacetic acid
  • TTHA triethylenetetraamine hexaacetic acid
  • the complexes which are suitable for application as near-IR luminescent long-lived diagnostic labels will, preferably, comprise a functional group which allows the attachment of the compound to an (immuno)reactant, e.g., an isothiocyanate, activated ester (such as N-hydroxysuccinimidyl ester), epoxide, or maleimide group for coupling to amino groups, an amino group for coupling to epoxides, a maleimide or halogenoacetamide group for coupling to thiol groups, a hydrazide group for coupling to aldehydes, and so on.
  • an (immuno)reactant e.g., an isothiocyanate, activated ester (such as N-hydroxysuccinimidyl ester), epoxide, or maleimide group for coupling to amino groups, an amino group for coupling to epoxides, a maleimide or halogenoacetamide
  • the erbium(lll) ion (Er 3+ ) is less preferred, because although it exhibits relatively long luminescence lifetimes ( ⁇
  • Um ), the luminescence is red-shifted to ⁇ * um max 1530 nm (see Tables I and II), which is unfavorable because water absorbs at this wavelength, and also in view of the expensive detectors which are required for detection at 1530 nm.
  • the lanthanides other than neodymium, ytterbium, and erbium do not give luminescence above the detection limit for excitation at 500 nm.
  • the advantages of the present invention are evident.
  • the near-IR luminescent complexes enable the design of low-cost detection devices.
  • visible light can be used which can be obtained from inexpensive light sources such as solid-state lasers, light-emitting diodes, or flash lamps.
  • inexpensive light sources such as solid-state lasers, light-emitting diodes, or flash lamps.
  • emission from ytterbium and neodymium silicon based detectors can be used, and expensive quartz optics are not required.
  • the near-IR luminescent complexes experience less interference from background signals originating from the sample solution and/or the detection optics, since visible excitation results in less scatter and less background luminescence. Moreover, the luminescence lifetime of the near-IR luminescent complexes is about two orders of magnitude shorter than that of the prior art Eu 3+ and Tb 3+ complexes. On the other hand, the luminescence lifetime of the near-IR luminescent ions is long enough to apply time-gated detection of their emission in order to further reduce the interference from background signals. The shorter lifetime of the near-IR luminescence is advantageous since the flux of luminescence photons is higher, and the number of background photons can be further decreased via the application of a narrower detection gate.
  • the reduced absorption of the visible radiation used for excitation makes it possible to apply the labels in opaque or strongly scattering matrices, for instance, in direct in situ measurements in body fluids and tissues.
  • the photochemical stability of the labels towards photo-oxidation is improved, since the triplet state of the sensitizer is efficiently depleted as a result of the energy transfer to the rare-earth ion.
  • the invention provides a method for detecting an analyte in a test sample, such as a body fluid or tissue of human, animal, bacterial, or vegetable origin.
  • the method is based on the reaction between the analyte and a specific binding partner for the analyte, and can be an immunoassay, an amplification assay, or hybridisation assay, such as NASBA and PCR.
  • the reaction product formed between the analyte and its specific binding partner is detected with a lanthanide ion-ligand complex according to the present invention, which is coupled to an (immuno)reactant.
  • This (immuno)reactant is a compound capable of binding, directly or indirectly, the analyte or the specific binding partner for the analyte.
  • the method can be performed according to different test formats, such as competitive or sandwich types of assays, which are well-known in the art.
  • sandwich type of assay the specific binding partner can be immobilized on a solid phase.
  • solid phases preferably polymer based core-shell latex particles or carrier materials (preferably porous carrier materials) are used. Examples are glass, membranes (e.g., nitrocellulose), and polymeric or ceramic (e.g., porous metaloxide) thin films.
  • a kit is provided for the qualitative or quantitative determination of an analyte jn a test sample, said kit comprising:
  • Measurement of the lanthanide ion-ligand complexes can be performed by excitation in the visible light range with inexpensive light sources such as solid-state lasers, light emitting diodes, or flash lamps, and detection of the emission by, for example, silicon based detectors.
  • inexpensive light sources such as solid-state lasers, light emitting diodes, or flash lamps
  • detection of the emission by, for example, silicon based detectors.
  • Such light sources and detectors can be used in combination with the device described above. It is therefore another object of the present invention to provide an apparatus comprising said kit in combination with a light source in the 400-1 OOOnm range, for instance a light emitting diode, and a detector which is suitable for detecting luminescence in the 800-1600 nm range, preferably the 800-1100 nm range, for instance a silicon or germanium based detector.
  • analytes can be measured, including antigens, antibodies, (glyco)proteins, peptides, oligo- nucleotides, nucleic acids, enzymes, haptens, and polysaccharides.
  • Specific binding partners for the analyte can be selected from antibodies, antigens, lectins, (oligo)nucleotides, receptors, and substrates.
  • the (immuno)reactant is a reactant which is capable of specifically binding to the analyte or specific binding partner for the analyte.
  • the (immuno)reactant can be selected from one of the analytes or specific binding partners as specified above. Specific examples are an oligonucleotide, an antibody, an anti-antibody, and protein A.
  • Examples of direct interaction type complexes sensitizers with complexing ability towards rare-earth ions.
  • Fluorexon was commercially obtained and used without further purification.
  • the lanthanide ions were added from stock solutions of YbCI 3 .6H 2 0, NdCI 3 .6H 2 0, and ErCI 3 .6H 2 0 in D 2 0 or H 2 0.
  • Fluorexon a well-known fluorescence indicator for Ca 2+ ions, was used to sensitize the near-IR (NIR) emission of trivalent ytterbium, neodymium, and erbium ions. Its absorption spectrum was similar to that of fluorescein, with an absorption maximum at 490 nm.
  • Solutions were prepared consisting of 5x10 "6 M of the fluorexon and an equimolar amount of lanthanide ions (Yb 3+ , Nd 3+ , or Er 3+ ) in D 2 0 at pD 7.
  • the pD was carefully controlled using an ISFET-based pH meter and concentrated solutions of DCI and NaOD.
  • the NIR luminescence excitation spectra of the respective Nd 3+ and Er 3+ complexes are identical to the spectrum of the fluorexon/Yb 3+ complex, and all match the corresponding absorption spectra, with an excitation maximum at 490 nm.
  • Methylthymol blue was commercially obtained and used without further purification.
  • the lanthanide ions were added from stock solutions of YbCI 3 .6H 2 0, NdCI 3 .6H 2 0 and ErCI 3 .6H 2 0 in D 2 0 and H 2 0.
  • Methylthymol blue (MTB) was demonstrated to be a luminescence sensitizing agent for ytterbium(lll), which emits light in a band around 1000 nm.
  • MTB does not sensitize Nd(lll). This was demonstrated in an experiment where both fluorexon (see Example 1) and MTB were brought into contact with Nd 3+ ions. From the absorption spectrum it was clear that both complexed MTB and fluorexon were present, but in the luminescence eexxcciittaattiioonn ssppeeccttrruumm ((eemmiisssiioonn aatt 11006600 nnrmr , the principal Nd 3+ emission line) only fluorexon sensitization was observed.
  • Chelates of the lanthanide ions of Nd(lll), Yb(lll), and Er(lll) were prepared by dissolving 2',7'-dichloro-4',5'-fluorexon-4-isothiocyanate and an equimolar amount of the lanthanide chloride in dry dimethylsulfoxide.
  • the NIR luminescence spectra obtained showed the sensitized excitation of the lanthanide ions with an excitation maximum of 508 nm.
  • the luminescence emission maxima were 880 nm, 1060 nm, and 1320 nm for Nd(lll) ion, 980 nm for Yb(lll) ion, and 1530 nm for Er(lll) ion.
  • the loading of the conjugate of a-hCG with the FXTC-Yb chelate was determined via an absorption measurement. For the three series of vials a loading varying from 5 to 15 FXTC-Yb labels per antibody was measured, depending on the amount of FXTC-Yb applied. A nitrocellulose membrane was spotted with 0.5 ⁇ l of a-hCG 147B and dried overnight. Subsequently, a mixture of 5000 IU (0.75 mg; 3.8 . 10 "13 mole) hCG and an excess of a-hCG 293 PA (2.0 . 10 "12 mole), loaded with 3.0.10 "11 mole of FXTC-Yb chelate, was incubated for 15 min.
  • the emitted light was focussed onto a liquid nitrogen cooled germanium detector (NorthCoast EO-817L, bias voltage 250V) through the same objective, the 510 nm long-pass dichroic, and a 830 nm long pass cut-off filter.
  • the signal was amplified with a lock-in amplifier (Stanford Instruments) and fed to a PC.
  • the scan showed a low background in the order of 1 V for the nitrocellulose and the negative (no hCG present) sample, and a clear signal, significantly enhanced to 8 V, for the positive sample with hCG (see Figure I).
  • the dichlorofluorexonisothiocyanate of Example 3 was dissolved in dry dimethylsulfoxide, and an equimolar amount of YbCI 3 .6H 2 0 was added. By measurement of the near-IR luminescence of the Yb(lll) ion, excited at 518 nm in the main absorption band of the dichlorofluorexon sensitizer unit, it was established that a chelate (FXTC-Yb) had been formed. To 50 ⁇ l of a solution of 1.24 mM amino-functionalized HIVQa oligonucleotide, consisting of 24 nucleotides (60 nmole), 0.5 ml of 0.1 M carbonate buffer (pH 9.0) were added.
  • the efficiency of labelling of the oligonucleotide with the FXTC-Yb chelate was determined via an absorption measurement.
  • For free oligonucleotide: ⁇ 280 280,000 l/mole. cm.
  • the labelling efficiency of oligonucleotide with FXTC-Yb was calculated to be about 100%.
  • a Whatman Anotec porous aluminum oxide membrane (with 200 nm diameter capillary pores) was purified ultrasonically for 0.5 h in MilliQ ultrapure water. Subsequently, the membrane was thoroughly rinsed with MilliQ water, after which the membrane was activated in a solution of 0.4 M nitric acid for 2 h, and thoroughly rinsed with MilliQ water. To ensure effective cleaning of the pores the membrane was put onto a glass filter, which was connected to a water jet filter pump. The rinsed membrane was dried for 1 h at 60°C under vacuum. A solution of 100 ⁇ l of 3-glycidyloxypropyltrimethoxy- silane in 7.5 ml of dried toluene was prepared and brought in contact with the activated membrane.
  • the membrane covered with the solution was incubated overnight at 60°C. Subsequently, the excess of solvent was removed, and the membrane was washed first with dried toluene, and subsequently with acetone. The with epoxy-groups modified Anotec membrane was dried at 50°C under vacuum.
  • This membrane was spotted with a concentration series of HIVQa-c oligonucleotides with amino end groups (the nucleotide sequence of which is complementary to that of the HIVQa oligonucleotides labelled with FXTC- Yb).
  • the oligonucleotides were incubated for 1 h.
  • the unbound oligo- nucleotides were remove by washing with 1 ml of wash fluid. Subsequently, the membrane was dried for 30 min at 37°C.
  • This membrane was brought into contact with 75 ⁇ l of the solution with HIVQa oligonucleotide labelled with FXTC-Yb.
  • the incubation time was 2 h at room temperature.
  • the unbound HIVQa-FXTC- Yb oligonucleotide was removed by washing with 10 ml of washing fluid.
  • the result of the hybridisation assay was established by scanning the membrane through the focal point of a 488 nm argon-ion laser, which illuminated a small area of the membrane through a mechanical chopper (385 Hz), a ⁇ 510 nm reflective dichroic beam splitter, positioned under 45° with respect to the laser beam in epifluorescence mode, and a 20x0.40 objective.
  • the emitted light was focussed onto a liquid nitrogen cooled germanium detector (NorthCoast EO-817L, bias voltage 250V), through the same objective, the 510 nm long-pass dichroic, and a 830 nm long pass cut-off filter.
  • the signal was amplified with a lock-in amplifier (Stanford Instruments) and fed to a PC. Examples of indirect interaction type complexes: sensitizers attached to compounds with complexing ability towards rare-earth ions.
  • Polyaminocarboxylic acids (such as ethylenediamine tetraacetic acid, EDTA, diethylenetriamine pentaacetic acid, DTPA, and triethylenetetraamine hexa- acetic acid, TTHA) form stable complexes with lanthanide ions.
  • sensitizer-modified derivatives of DTPA have been obtained by reaction of its commercially available dianhydride (DTPAA) with an amine.
  • Dye derivatives which absorb in the visible part of the spectrum like fluorescein and eosin, have been used as amine.
  • the products thus obtained form stable complexes with lanthanides (Yb 3+ , Nd 3+ , Er 3+ ), which give intense NIR luminescence upon irradiation with visible light, even in aqueous solution.
  • Example 6 DTPAA and 5-aminofluorescein were commercially obtained and used without further purification.
  • the lanthanide ions were added from stock solutions of YbCI 3 .6 H 2 0, NdCI 3 .6 H 2 0 and ErCI 3 .6 H 2 0 in D 2 O and H 2 0.
  • TEAAc triethylammoniumacetate
  • MeCN acetonitrile
  • the NIR luminescence spectra were recorded with an excitation wavelength of 490 nm, the absorption maximum of fluorescein. They all showed the sensitized lanthanide luminescence. Spectra obtained for the complexes clearly showed the sensitizing properties of fluorescein: the emission of trivalent ytterbium, neodymium, and erbium at their typical emission wavelengths could be observed when excited at 490 nm, the absorption maximum of fluorescein. In addition, the luminescence excitation spectrum of the Nd(lll) emission (at 1060 nm) and the ytterbium emission (at 980 nm) have been recorded and were an excellent match of the absorption (and excitation) spectrum of fluorescein.
  • DTPAA was commercially obtained and used without further purification. 5- aminoeosin was commercially purchased.
  • the lanthanide ions were added from stock solutions of YbCI 3 .6 H 2 0, NdCI 3 .6 H 2 0 and ErCI 3 .6 H 2 0 in D 2 0 and H 2 0.
  • Steady state luminescence measurements were performed on a PTI Alphascan spectrofluorimeter, using a 75-W quartz-tungsten-halogen lamp followed by a SPEX 1680 double monochromator for excitation and a PTI 0.25-m single monochromator for separation of the emitted light, detected under an angle of 90°.
  • the emitted light was converted into an electric signal with a Northcoast 817L liquid nitrogen cooled Germanium detector.
  • a lock-in amplifier (SRS530) was applied; the excitation light was modulated at 70 Hz with an optical chopper.

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Abstract

L'invention concerne un procédé pour la détection d'un analyte dans un échantillon d'essai par une réaction de liaison spécifique dans l'analyte, un partenaire de liaison spécifique pour l'analyte, et un (immuno)réactif pourvu d'un marqueur. Ce procédé se caractérise en ce que le marqueur est un complexe ligand-ion de lanthanide où l'ion de lanthanide est un ion (Nd3+) de neodymium (III), un ion (Yb3+) d'ytterbium (III) ou un ion (Er3+) d'erbium et le ligand comprend, ou est en contact avec une fraction de sensibilisation dont l'absorption se situe dans la zone de 400 à 1000 nm, et de préférence, dans la zone de 400 à 800 nm. En outre, un kit de diagnostique est décrit ainsi qu'un procédé pour détecter un analyte dans une matrice présentant un intérêt biomédical à l'aide d'un oligonucléotide, un antigène, ou un anticorps fixé à un matériau, de préférence, un latex de coeur-coquille ou à des sites de liaison spécifiques où l'antigène ou l'anticorps est marqué avec le complexe ligand-ion de lanthanide et amené en contact avec l'analyte, après quoi l'analyte avec le complexe d'ions de lanthanide est retiré. L'échantillon obtenu est ainsi irradié à la lumière dans la zone de 400 à 1000 nm, et la lumière émise par cet échantillon est détectée lorsque cet analyte est présent dans la matrice présentant un intérêt biomédical.
PCT/EP1998/001287 1997-03-03 1998-02-28 Complexes de ligands-ions de neodymium (iii), ytterbium (iii) ou erbium (iii) WO1998039654A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU68284/98A AU6828498A (en) 1997-03-24 1998-02-28 Diagnostic neodymium(iii), ytterbium(iii), or erbium(iii) ion-ligand complexes
EP98913667A EP0968424A1 (fr) 1997-03-03 1998-02-28 Complexes de ligands-ions de neodymium (iii), ytterbium (iii) ou erbium (iii)
US09/380,336 US6951760B2 (en) 1997-03-03 2000-02-04 Diagnostic neodymium(III), ytterbium(III), or erbium(III) ion-ligand complexes

Applications Claiming Priority (4)

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EP97200615.9 1997-03-03
EP97200615 1997-03-03
US4235497P 1997-03-24 1997-03-24
US60/042,354 1997-03-24

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WO1998039654A3 WO1998039654A3 (fr) 2000-01-06

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6902904B2 (en) * 2001-08-27 2005-06-07 Pharmanetics Incorporated Coagulation assay reagents containing lanthanides
US7160732B2 (en) * 2000-07-07 2007-01-09 Massachusetts Institute Of Technology Fluorescein-based metal sensors, and methods of making and using the same

Family Cites Families (5)

* Cited by examiner, † Cited by third party
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US5587394A (en) * 1991-03-28 1996-12-24 The University Of Toledo Production and use of diels alder adducts of vinyl porphyrins, of metal complexes thereof, and of compositions containing such adducts and complexes
US5312922A (en) * 1992-04-06 1994-05-17 Nordion International Inc. Europium and terbium chelators for time-resolved fluorometric assays
US5622821A (en) * 1994-06-29 1997-04-22 The Regents Of The University Of California Luminescent lanthanide chelates and methods of use

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7160732B2 (en) * 2000-07-07 2007-01-09 Massachusetts Institute Of Technology Fluorescein-based metal sensors, and methods of making and using the same
US6902904B2 (en) * 2001-08-27 2005-06-07 Pharmanetics Incorporated Coagulation assay reagents containing lanthanides

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