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WO1999031513A1 - Procede et composition pour caracteriser un echantillon contenant un lipide echantillon, et trousse de dosage - Google Patents

Procede et composition pour caracteriser un echantillon contenant un lipide echantillon, et trousse de dosage Download PDF

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Publication number
WO1999031513A1
WO1999031513A1 PCT/US1998/026559 US9826559W WO9931513A1 WO 1999031513 A1 WO1999031513 A1 WO 1999031513A1 US 9826559 W US9826559 W US 9826559W WO 9931513 A1 WO9931513 A1 WO 9931513A1
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metal
lipid
ligand complex
fluorescent
sample
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Joseph R. Lakowicz
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    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention is in the field of characterizing a sample containing a sample lipid.
  • U.S. Patent No. 5,322,794 discloses coronene- labeled lipids and methods for characterizing lipids .
  • coronene-labeled lipids can only exhibit single lifetimes.
  • Fluorescent liposomes have been used for immunoassays . Fluorescein-phosphatidyl ethanolamine has been used in heterogeneous immunoassays. Liposomes are useful in high sensitivity assays because many fluorophores can be bound to one liposome. However, fluorophore-derivatized liposomes are sensitive to concentration quenching.
  • a method of characterizing a sample containing a sample lipid includes the step of adding a metal-ligand complex lipid probe to a lipid-containing sample to form a mixture containing labeled sample lipid.
  • the labeled sample lipid is comprised of the sample lipid labeled with the metal-ligand complex lipid probe.
  • the mixture is exposed to an exciting amount of electromagnetic light energy which causes the labeled sample lipid to emit fluorescent light. The emitted light is measured.
  • composition for characterizing a lipid containing sample includes a fluorescent metal-ligand complex coupled to a lipid.
  • an assay kit includes a fluorescent metal-ligand complex and instructions for coupling the fluorescent metal-ligand complex with a lipid.
  • FIG. 1 is a graph depicting metal-ligand complex lipid probes made with rhenium, ruthenium, and osmium that have different emission wavelengths.
  • FIG. 2 shows long-lifetime rhenium carbonyl complexes that can be attached to lipids.
  • FIG. 3 graphically depicts absorption and emission spectra of Ru-PE and Ru-PE 2 in DPPG vesicles at 20°C, and the excitation anisotropy spectra of the parent compounds of Ru (bpy) 2 (mcbpy) +1 and Ru (bpy) 2 (dcbpy) in glycerol/water at -55°C.
  • FIG. 4 graphically depicts the frequency-domain intensity decays of MLC-PE and MLC-PE 2 in DPPG vesicles at various temperatures.
  • FIG. 5 graphically depicts temperature dependent steady state anisotropies of DPPG vesicles labeled with Ru-PE or Ru-PE 2 .
  • FIG. 6 shows structures of two MLC lipid probes: Ru (bpy) 2 (mcbpy) -PE (Ru-PE) and Ru (bpy) 2 (dcpby) -PE 2 (Ru- PE,) .
  • FIG. 7 graphically depicts frequency-domain anisotropy decays of Ru-PE in DPPG vesicles.
  • FIG. 8 graphically depicts frequency-domain anisotropy decays of Ru-PE 2 in DPPG vesicles.
  • a method for characterizing a sample lipid by measuring the light emitted by the sample lipid labeled with a metal-ligand complex lipid probe .
  • a composition which includes a fluorescent metal-ligand complex, for characterizing a lipid containing sample.
  • an assay kit which includes a metal-ligand complex and instructions for coupling the metal-ligand complex with a lipid.
  • Metal-ligand complex lipid probes are particularly useful because they display decay times longer than any available lipid probe. Additionally, the use of metal- ligand complexes provides probes with a range of lifetimes. This is in contrast to the single lifetime available from coronene-labeled lipids.
  • Metal-ligand complex lipid probes allow increased high sensitivity detection by gating off the prompt autofluorescence from the samples.
  • An advantage of using metal-ligand complex lipid probes in immunoassays is that metal-ligand complexes are less sensitive to the concentration quenching that occurs with fluorescein.
  • Lipid probes in accordance with the invention that contain covalently linked metal-ligand complexes have been synthesized.
  • the inventive long lifetime metal- ligand complex lipid probes can be used to characterize lipid-containing samples . The change in fluorescent light emission of the probe and fluorescent light emission of the sample lipid labeled with the metal- ligand complex lipid probe is utilized to characterize the lipid.
  • the intensity of the emitted light can be measured and used to characterize the sample lipid.
  • the lifetime of the emitted light can also be measured and used to characterize the sample lipid. If linearly polarized light energy is used to excite the mixture of the sample lipid labeled with the metal-ligand complex lipid probe, then the lipid is characterized by measuring the polarization of the emitted light.
  • Metal-ligand complex lipid probes can be used to characterize the membrane dynamics of lipid-containing samples. Metal-ligand complex lipid probes can be used to measure the rotational hydrodynamics of phospholipid vesicles . These probes can be used to measure microsecond rotational motions of lipid vesicles or within lipid membranes.
  • Metal-ligand complex lipid probes can also be used to measure two-dimensional diffusion in membranes.
  • the probe lipid can be any suitable lipid including, but not limited to, phospholipids and phosphatidyl ethanolamine .
  • Metal-ligand complex lipid probes can also be used in clinical assays based on liposomes. Metal-ligand complex lipid probes can be used in homogeneous and heterogeneous assays. Metal-ligand complex lipid probes can be used in homogeneous assays because of the long lifetime of the metal-ligand complexes, which allows gating and using fluorescence polarization or anisotropy which is independent of intensity. Examples of sample and probe lipids include, but are not limited to, liposomes and phospholipids, respectively.
  • the metal-ligand complex lipid probes of the present invention may be used in place of the known fluorophores in any suitable assay method.
  • metal-ligand complexes which display luminescence, including complexes containing Co, ' Cr, Cu, Mo, Ru, Rh, W, Re, Os, Ir, or Pt .
  • transition metal complexes especially those with Ru, Os, Re, Rh, Ir, W or Pt, can be used in accordance with the invention.
  • the metal in the metal- ligand complex is particularly preferably selected from the group consisting of ruthenium, osmium, and rhenium.
  • a suitable ligand in the metal-ligand complex can be polypyridine, bipyridine, or a related compound, and the ligand can contain a reactive group commonly used for linkage to biological molecules, such as a N- hydroxysuccinimide ester of a carboxylic acid, haloacetyl groups, maleimides, sulfonyl chlorides, and isothiocyanates .
  • Other ligands for such metal-ligand complexes are bipyrazyl, phenanthroline, and related substituted derivatives, or inorganic ligands such as CO, Cl, nitrile and isonitrile.
  • Preferred metal-ligand complexes include [Ru(2,2'- bipyridine) 2 (4-carboxy-4 ' -methyl-2, 2 ' -bipyridine) ] ; [Ru (2,2' -bipyridine) 2 (4, 4 ' -dicarboxy-2, 2 ' -bipyridine) ] ; [Re (2, 9-dimethyl-4,7-diphenyl-l, 10- phenanthroline) (CO) 3 (monosubstituted pyridine)]*; [Re (3, 4,7, 8-tetramethyl-l, 10- phenanthroline) (CO) 3 (monosubstituted pyridine)] + ; [Re(5- phenyl-1, 10-phenanthroline) (CO) 3 (monosubstituted pyridine) ] + ; [Re (4 , 7-diphenyl-l, 10- phenanthroline) (CO) 3 (monosubstituted pyridine)
  • One embodiment of the present invention utilizes phospholipid analogues of phosphatidyl ethanolamine which contain a ruthenium metal-ligand complex covalently bound to the amino group.
  • a detailed description of the synthesis and fluorescence characterization is' ' disclosed in Li, L., Szmacinski, H., and Lakowicz, J.R., "Synthesis and Luminescence
  • a metal-ligand complex lipid probe is utilized in a fluorescence polarization immunoassay of a high molecular weight antigen.
  • the present invention thus can be incorporated into the method disclosed in Terpetschnig, E., Szmacinski, H., and Lakowicz, J.R., "Fluorescence Polarization Immunoassay of a High- Molecular-Weight Antigen Based on a Long-Lifetime Ru- Ligand Complex," Analytical Biochemistry 227:140-47 (1995), incorporated herein by reference.
  • Ru (bpy) 2 (mcbpy) (360 mg) and 51 mg of N- hydroxysuccinimide ( ⁇ HS) were dissolved in 1.2 ml of acetonitrile at room temperature.
  • the synthesis of Ru (bpy) 2 (mcbpy) is described in Szmacinski, H., Terpetschnig, E., and Lakowicz, J.R. (1996) Biophys . Chem.. in press.
  • N, N'-Dicyclohexylcarbodiimide (DCC, 120 mg) was then added. The mixture was sealed and stirred for a few hours. The formed precipitate was removed by filtration through a syringe filter (Nylon Acrodish, 0.45-um pore size).
  • Ru (bpy) 2 (mcbpy) -NHS 120 mg was dissolved in 4.5 ml of dry DMF and slowly added to a stirring solution of PE 980 mg in 10 ml of CHC1 3 ) and triethylamine (6.0 ml) under an argon atmosphere. The mixture was stirred for 20 hours in the dark. The solvents were removed under vacuum and the product was redissolved in 2.5 ml of CHCl 3 /MeOH (2/1, v/v) . The pure Ru-PE was obtained by TLC on K6F silica gel plates using CHCl 3 /MeOH/H 2 0
  • Ru-PE 2 was prepared in analogy to Ru-PE.
  • the purification by TLC was repeated twice to obtain pure Ru-PE 2 , with a final, yield of about 2.3%.
  • Emission spectra were recorded on a SLM AB2 spectrofluorometer .
  • the frequency-domain instrumentation (ISS) was used for measurements of the fluorescence intensity and anisotropy decays.
  • the excitation source was a CW air-cooled argon ion laser (543-AP, Omnichrome) .
  • the laser was amplitude modulated by the electrooptical low-frequency modulator (K2.LF from ISS) and was tuned at 488 nm as the excitation wavelength.
  • K2.LF electrooptical low-frequency modulator
  • a 610-nm cutoff filter was also used to isolate the emission of Ru complexes.
  • the frequency-domain intensity and anisotropy data were fit to single and double-exponential decay laws.
  • g i r 0 is the amplitude of the anisotropy decaying with the correlation time ⁇ j
  • r 0 is the anisotropy in the absence of rotational diffusion.
  • the parameter values were recovered as described in Lakowicz, J.R., Gratton, E., Laczko,
  • lipids with a Ru- lipid/DPPG mole ratio ranging from 1:20 to 1:100 were dissolved in CHCl 3 /MeOH (95/5, v/v) .
  • the lipid- containing solution was kept in a water bath at a constant temperature (55°C) , and the solvent was removed by a stream of argon.
  • Vesicles were prepared by sonication in 10 mM Tris and 50 mM KCl, pH 7.5, at a final lipid concentration of 2 mg/ml.
  • the DPPG vesicles in the absence of Ru-lipid did not display significant signals ( ⁇ 1.5%) under the present experimental conditions. Unless indicated otherwise, all measurements were performed in the presence of dissolved oxygen from equilibrium with the air.
  • the anisotropies of Ru-PE 2 are consistently larger than those of Ru-PE, as expected from the higher fundamental anisotropy of Ru (bpy) 2 (dcbpy) and possibly due to less segmental motion of the MLC covalently bound to two PE molecules.
  • the anisotropy decays display both a short ⁇ - 100 NS) and long [ ⁇ 1.5 ⁇ s) correlation time. These longer correlation times are consistent with those expected for overall rotational diffusion of phospholipid vesicles with diameter from 200 to 300 A: For instance, vesicles with a diameter of 250 A are expected to display a correlation time near 2020 ns
  • lipid probes Another important characteristic of a lipid probe is the extent of labeling possible without spectral changes due to probe-probe interactions.
  • Metal-ligand complex lipid probes can be regarded as the first of many such probes with different spectral properties. For instance, it is known that the decay time of ruthenium metal-ligand complexes can be increased to several microseconds by the use of diphenyl-phenanthroline ligands, Lakowicz, J.R., Murtaza, Z., Jones, W.E., Kim, K., and Szmacinski, H. (1996) J. Fluoresc. 6:245-249; Demas, J.N., Harris, E.W., and McBridge, R.P. (1977) J. Am. Chem. Soc .
  • Rhenium complexes also display polarized emission
  • Metal-ligand complex lipid probes can be designed with a range of emission wavelengths, lifetimes, and quantum yields, and can have wide ranging applications for studies of the dynamics of model and cell membranes .

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Abstract

On décrit un procédé pour caractériser un échantillon contenant un lipide échantillon. Le procédé consiste à ajouter un complexe métal-ligand à un échantillon contenant du lipide pour former un mélange contenant un lipide échantillon marqué. Ce lipide échantillon marqué inclut le lipide échantillon marqué avec la sonde lipidique du complexe métal-ligand. Le mélange est mis en présence d'une quantité d'énergie lumineuse électromagnétique suffisante pour exciter le lipide échantillon marqué et l'amener à émettre une lumière fluorescente. Cette lumière émise est mesurée. Une composition pour caractériser un échantillon contenant du lipide inclut un complexe métal-ligand fluorescent associé à un lipide. On décrit une trousse de dosage incluant un complexe métal-ligand fluorescent et des instructions pour associer ledit complexe avec un lipide.
PCT/US1998/026559 1997-12-15 1998-12-14 Procede et composition pour caracteriser un echantillon contenant un lipide echantillon, et trousse de dosage Ceased WO1999031513A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8216582B2 (en) 2006-06-23 2012-07-10 Alethia Biotherapeutics Inc. Polynucleotides and polypeptide sequences involved in cancer
US8580257B2 (en) 2008-11-03 2013-11-12 Alethia Biotherapeutics Inc. Antibodies that specifically block the biological activity of kidney associated antigen 1 (KAAG1)
US8937163B2 (en) 2011-03-31 2015-01-20 Alethia Biotherapeutics Inc. Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
EP3221706A4 (fr) * 2014-11-21 2018-05-23 University Of South Australia Procédés et produits utilisables en vue du marquage de lipides
US11084872B2 (en) 2012-01-09 2021-08-10 Adc Therapeutics Sa Method for treating breast cancer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4708933A (en) * 1984-06-12 1987-11-24 Leaf Huang Immunoliposome assay-methods and products
US5310687A (en) * 1984-10-31 1994-05-10 Igen, Inc. Luminescent metal chelate labels and means for detection
US5660991A (en) * 1994-10-28 1997-08-26 Lakowicz; Joseph R. Long lifetime anisotropy (polarization) probes for clinical chemistry, immunoassays, affinity assays and biomedical research

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4708933A (en) * 1984-06-12 1987-11-24 Leaf Huang Immunoliposome assay-methods and products
US5310687A (en) * 1984-10-31 1994-05-10 Igen, Inc. Luminescent metal chelate labels and means for detection
US5660991A (en) * 1994-10-28 1997-08-26 Lakowicz; Joseph R. Long lifetime anisotropy (polarization) probes for clinical chemistry, immunoassays, affinity assays and biomedical research

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI L., ET AL.: "LONG-LIFETIME LIPID PROBE CONTAINING A LUMINISECENT METAL-LIGAND COMPLEX.", BIOSPECTROSCOPY., WILEY, NEW YORK, NY, US, US, vol. 03., 1 January 1997 (1997-01-01), US, pages 155 - 159., XP002916604, ISSN: 1075-4261, DOI: 10.1002/(SICI)1520-6343(1997)3:2<155::AID-BSPY8>3.0.CO;2-6 *
LI L., SZMACINSKI H., LAKOWICZ J. R.: "SYNTHESIS AND LUMINESCENCE SPECTRAL CHARACTERIZATION OF LONG- LIFETIME LIPID METAL-LIGAND PROBES.", ANALYTICAL BIOCHEMISTRY., ACADEMIC PRESS INC., NEW YORK., vol. 244., 1 January 1997 (1997-01-01), NEW YORK., pages 80 - 85., XP002916605, ISSN: 0003-2697, DOI: 10.1006/abio.1996.9869 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8216582B2 (en) 2006-06-23 2012-07-10 Alethia Biotherapeutics Inc. Polynucleotides and polypeptide sequences involved in cancer
US8580257B2 (en) 2008-11-03 2013-11-12 Alethia Biotherapeutics Inc. Antibodies that specifically block the biological activity of kidney associated antigen 1 (KAAG1)
US9855291B2 (en) 2008-11-03 2018-01-02 Adc Therapeutics Sa Anti-kidney associated antigen 1 (KAAG1) antibodies
US8937163B2 (en) 2011-03-31 2015-01-20 Alethia Biotherapeutics Inc. Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
US9393302B2 (en) 2011-03-31 2016-07-19 Alethia Biotherapeutics Inc. Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
US9828426B2 (en) 2011-03-31 2017-11-28 Adc Therapeutics Sa Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
US10597450B2 (en) 2011-03-31 2020-03-24 Adc Therapeutics Sa Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
US11084872B2 (en) 2012-01-09 2021-08-10 Adc Therapeutics Sa Method for treating breast cancer
EP3221706A4 (fr) * 2014-11-21 2018-05-23 University Of South Australia Procédés et produits utilisables en vue du marquage de lipides

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