EP0807254A1 - Lumineszierende sonden zum nachweis von proteinen - Google Patents

Lumineszierende sonden zum nachweis von proteinen

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Publication number
EP0807254A1
EP0807254A1 EP96941407A EP96941407A EP0807254A1 EP 0807254 A1 EP0807254 A1 EP 0807254A1 EP 96941407 A EP96941407 A EP 96941407A EP 96941407 A EP96941407 A EP 96941407A EP 0807254 A1 EP0807254 A1 EP 0807254A1
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European Patent Office
Prior art keywords
lanthanide
protein
chelate
target
domain
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EP96941407A
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English (en)
French (fr)
Inventor
Wayne F. Patton
David Shepro
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Boston University
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Boston University
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to methods and compositions for reversible binding to and detection of targets using luminescent lanthanide chelates, and lanthanide-chelate-target complexes
  • the targets may be proteins or nucleic acids
  • the invention also relates to kits which comprise lanthanide chelates or lanthanide-chelate-target complexes
  • solution-based methods such as the Coomassie brilliant blue and pyrogallol red-molybdate assays measure shifts in the wavelength maximum of dye absorbance upon formation of a dye-protein complex
  • Many solution-based colorimetric total protein labels are adaptable to solid-phase formats Since the protein is specifically bound to a membrane or other solid support, contaminants are readily washed away Therefore, solid-phase protein assays are less susceptible to interfering agents commonly found in the sample, such as 2-mercaptoethanol, Tris, salts and detergents Total protein quantification is easily achieved directly on the membrane support by densitometry using instrumentation such as a flat-bed scanner or CCD camera Alternatively, the stains can be eluted from membranes and quantified using a spectrophotometer Since the assays are performed on a solid-phase support, both small and large sample volumes are readily accommodated Very dilute protein solutions are easily concentrated on the membrane by repeated application of samples The colored product is usually stable, so there are no stringent time requirements for completion ofthe assays Analysis is
  • Chemiluminescence is a special type of fluorescence whereby light is produced as the result of a chemical reaction When this process is performed through biological means it is referred to as bioluminescence
  • the firefly enzyme luciferase a well-known example, produces light upon the oxidation of luciferin
  • Fluorescence is the emission of light as a molecule returns from an excited state to the ground state Phosphorescence is similar to fluorescence, but the excited molecule first undergoes a transition to a long-lived excited state It then returns slowly to the ground state over several seconds and light is emitted Phosphorescence and fluorescence are often not carefully distinguished in the technical literature, however, and many phosphorescing labels are referred to as fluorescent However, in reality a given molecule may both fluoresce and phosphoresce, making the distinction somewhat academic Examples of organic luminescent labels include coumarin, fluorescamine, rhodamine, erythrosine, eosine, dansyl chloride, and their derivatives
  • the rare earth elements comprise the cerium and yttrium subgroups Although yttria is not a rare earth element, it is typically found associated with the rare earths and can only be separated with difficulty Typical sources ofthe rare earths include monazite, bastnasite and related fluocarbonate materials as well as minerals ofthe yttrium group. Rare earth elements also occur as fission products of uranium and plutonium, and are the only source of promethium
  • the luminescence of a metal chelate is, in many ways, analogous to the process of photosynthesis.
  • the chlorophyll molecules and accessory pigments are clustered into light-harvesting antenna complexes in the lamellae ofthe thykaloid discs ofthe chloroplasts
  • Light is absorbed by a molecule of chlorophyll or one ofthe accessory pigments, which leads to an excited state
  • the excited state is passed from one pigment molecule to another until it reaches the reaction center.
  • a special chlorophyll molecule converts the energy ofthe excited state to chemical energy in the form of ATP or NADPH
  • the metal ion can be thought of as the reaction center.
  • the organic chelator molecules act as the light- harvesting antenna for the metal ion, but instead of converting the absorbed light into chemical energy, the metal ion re-emits the energy as light
  • the chelator molecules efficiently absorb light and pass it to the metal ion This excites electrons from the F shell to the D shell and, upon subsequent decay to the ground state, longer wavelength light is released
  • the nature of the chelate determines the efficiency and wavelength of the absorbed light, but the emission wavelength is determined by the metal ion itself and is not greatly affected by the antenna molecules
  • Metals used in this fashion include the lanthanides, europium, terbium, samarium, gadolinium, and dysprosium
  • the lanthanide chelates, especially those of europium and terbium, are well suited as biological labels
  • the absorbance ofthe chelates can be very strong and varies with the chelate selected, but generally the excitation maximum is within the UV (200-360 nm), allowing the
  • the quantum yield of lanthanide chelates is often less than that of organic luminescent labels, the lanthanides emit at relatively long wavelengths where the background biological fluorescence is low
  • the long Stokes shift makes it easy to detect emitted light without flooding the detector with excitation wavelength light and the long lifetime of the luminescence (50-1000 ⁇ sec) allows the measurement of signal after the short duration biological fluorescence has decayed to background For all of these reasons, the signal to noise ratio is high
  • iron chelates have been utilized as labels that produce colored complexes when complexed with proteins (Patton, W F , et al (1994) Anal. Biochem. 220 324-335)
  • This total protein labeling technique was shown to be compatible with microsequencing and immunodetection, but increased sensitivity and a broader dynamic range is still desired
  • the iron chelate technique as described is incompatible with detection of protein in semi-solid materials such as gels
  • Certain lanthanide chelates have been used as biological labels in limited instances
  • a generic description of the lanthanide chelates comprises a triplet sensitizer nucleus and three heteroatom containing groups (U S Pat No 4,637,988) This structure allows for the transfer of energy from the chelate to the lanthanide
  • Time resolved fluorometry has been used in combination with some lanthanide chelates (U S Pat No 4,374,120)
  • This technique utilizes an EDTA- like molecule that is covalently bound to a target molecule, such as an antibody
  • a target molecule such as an antibody
  • the bifunctional EDTA-like molecule allows the binding of both the antibody and the lanthanide
  • the resulting structure is further complexed with a di-ketone or di ⁇ hydroxy molecule which functions to absorb light
  • the 1/1/1 (antibody- EDTA/Eu/diketone) complex is then detected by time resolved fluorometry
  • the method is suitable for sandwich immunoassays where the secondary antibody is conjugated to an EDTA-like molecule which is then capable of complexing with a lanthanide chelate
  • the technique is generally unsuitable for most biochemical analyses, including microsequencing
  • a selected substrate is enzymatically and reversibly transformed into a compound that will complex with lanthanide and phosphoresce
  • alkaline phosphatase and 5-fluorosaIicylate phosphate compounds Alkaline phosphatase converts 5-fluorosalicylate phosphate into 5-fluorosalicylate which combines with europium and phosphoresces when excited at the appropriate wavelength
  • alkaline phosphatase converts 5-fluorosalicylate phosphate into 5-fluorosalicylate which combines with europium and phosphoresces when excited at the appropriate wavelength
  • chelates have been tested in enzymatic, time resolved, fluorometric immunoassays (U S Pat No 5,312,922)
  • a hydroxyl group on a chelate may be converted to a phosphate ester or galactoside for use with the enzymes alkaline phosphatase or ⁇ -galactosidase Esters are enzymatically treated to be
  • the present invention overcomes the problems and disadvantages associated with current labeling techniques and provides novel methods for the detection and isolation of molecular targets such as proteins and nucleic acids
  • One embodiment ofthe invention is directed to lanthanide chelate comprising a lanthanide and a ligand wherein the ligand has a first domain that binds to the lanthanide, a second domain that reversibly binds to a target, and a third domain that absorbs UV radiation
  • Typical lanthanide chelates comprise an element of europium, terbium, samarium, gadolinium or dysprosium, and a ligand such as bathophenanthroline disulfonic acid Targets may be proteins or nucleic acids, or fragments or constituents thereof
  • Another embodiment of the invention is directed to lanthanide- chelate-target complexes comprising a lanthanide, a ligand and a target, wherein the ligand has a first domain that binds to the lanthanide, a second domain that reversibly binds to the target, and a third domain that absorbs UV light
  • Targets may be, for example, proteins, peptides, amino acids, nucleic acids, nucleotides or fragments or modifications thereof
  • Another embodiment of the invention is directed to methods for detecting a target in a sample
  • the sample to be screened is contacted with a lanthanide chelate wherein the lanthanide chelate reversibly binds to the target
  • This sample is illuminated with electromagnetic radiation, such as ultraviolet radiation, and any radiation emitted is detected and can be quantified
  • Samples which can be tested include patient and environmental samples Because the lanthanide-chelate- target interaction is a non-covalent one, the reaction is fully reversible Thus, the lanthanide chelate can be removed and the target utilized for procedures such as sequencing
  • Another embodiment of the invention is directed to methods for detecting protein Protein is contacted with a lanthanide chelate wherein the chelate has a first domain that binds to the lanthanide, a second domain that reversibly binds to protein, and a third domain that absorbs UV light Binding is non-covalent and fully reversible Bound protein is illuminated with UV and the emitted phosphorescence is detected
  • Another embodiment of the invention is directed to a multi-label immunodetection method whereby total protein and one or more target proteins can be sequentially detected
  • a sample is contacted with a primary antibody that specifically binds to a target protein
  • the sample is contacted with a second antibody that specifically binds to the primary antibody, wherein the second antibody has a polyamine-tag which functions to increase the stability of lanthanide chelate binding at increased pH.
  • the sample is contacted with a lanthanide chelate at a first, acidic pH and the phosphorescence of total protein in the sample is measured.
  • Antibodies may be monoclonal or polyclonal, but preferably the primary antibody is monoclonal and the secondary antibody is polyclonal
  • Another embodiment of the invention is directed to methods for precipitating a target protein from a sample
  • the sample containing target protein is contacted with a lanthanide chelate wherein the lanthanide chelate reversibly binds to the protein
  • Target protein can be collected by, for example, filtration or centrifugation.
  • Another embodiment of the invention is directed to methods for isolating a target molecule from a sample without the requirement of covalent modification of the target.
  • the sample is contacted with a lanthanide chelate and the lanthanide chelate reversibly binds to the target.
  • the mixture is illuminated with UV to produce phosphorescence Phosphorescing targets are identified and can be isolated from other substances.
  • kits for the detection of a target comprising a lanthanide chelate or a lanthanide-chelate-protein complex Targets may be detected in any biological samples, such as samples from patients, animals, cultures, or from the environment, such as in soil or water samples
  • the kit may further comprise a binding buffer and an elution buffer
  • FIG. 1 Multi-Detection Immunoassay A schematic representation of a multi-detection immunoassay (A ) All proteins are detected at low pH and (B ) sequential detection of polyamine-tagged protein is achieved by increasing the pH 1 is the europium complex; 2 is the amine-labeled secondary antibody, 3 is the primary antibody, 4 is the protein, and 5 is the membrane support
  • FIG. 7 Binding to Polyamino Acids Binding of bathophenanthroline disulfonate-europium to various homopolymers and heteropolymers of amino acids to determine the effect of amino acid side chains on binding
  • the present invention is directed to chemicals, chemical complexes and methods for labeling targets such as proteins, nucleic acid, and derivatives, and to kits which comprise these chemicals and chemical complexes
  • a highly sensitive, broadly applicable method for protein detection is presently needed. This method should also be compatible with typical analytical processes such as microsequencing, immunoassay, enzymatic assays, mass spectrometry, carbohydrate analysis and Western blotting Criteria for the labeling of a protein with a sensitive and specific label include high affinity for the protein, low affinity for other biological materials as well as common laboratory reagents, rapid and straightforward labeling techniques and application conditions.
  • the label should also be compatible with a wide range of electrophoretic matrices and have a large quantum yield for maximal sensitivity An emission maximum in the 500- 600 nm region of the spectrum avoids interference from common biological fluorophores It should also have quantifiable binding over a broad range of total protein (a broad dynamic range) The label should have an ability to amplify for increased sensitivity, a reasonable cost, low toxicity and be readily available Labels should also be completely reversible so that other specific stains or biochemical assays may be employed
  • Lanthanide chelates of this invention meet these requirements as labeling tools for biological and other target molecules
  • Lanthanide chelates comprise an element selected from the lanthanide series and a chelate (or ligand or chelator) that will bind to a metal ion
  • the chelate comprises a ligand that reversibly attaches to a target Attachments may be through hydrophobic, hydrophilic, Van der Waals, and ionic or other non-covalent interactions
  • the chelate absorbs energy from UV radiation and transmits this energy to the lanthanide which, in response, phosphoresces.
  • Ligands contain a first domain which binds to the lanthanide, and preferably contain side chains that are combinations of ketone, carboxyl, hydroxyl or pyridine groups.
  • a second domain reversibly binds to protein and should consist of one or more anionic residues for electrostatic interaction with protonated amines of proteins (the negative charge of the second ligand domain interacts with the positive charge ofthe protonated amine).
  • Groups that function in this regard include sulfonate, sulfate, phosphonate and phosphate groups.
  • Carboxylate groups are less appropriate for this pu ⁇ ose Aromatic or heteroaromatic functionalities also enhance binding avidity to targets such as proteins by hydrophobic interactions.
  • a third domain absorbs the excitation light which is then transferred to the lanthanide ion, which subsequently emits the light at a longer wavelength
  • This domain preferably contains 5- or 6-member monocyclic or polycyclic, aromatic or heteroaromatic (containing oxygen and/or nitrogen and/or sulfur) ring structures, a necessity for chromophores.
  • the chelating groups may bind and transfer the excitation energy to the surface of a lanthanide colloid to form the luminescent complex.
  • Chelate and/or target binding sites can be incorporated into the ring structure ofthe chromophore or, alternatively, distinct chelate, chromophore and target binding sites can be engineered, allowing for separate optimization of each domain
  • Lanthanide chelates may also be clathrate compounds which are inclusion complexes wherein molecules of one substance are completely enclosed within another Chelates may also contain three functional domains distributed among two or more molecules For example, one molecule may contain a chromophore and lanthanide-binding domain while the other contains lanthanide-binding and protein- binding domains Resulting complexes between the lanthanide and the two chelators would contain all three functional domains
  • Chelates that function in this regard include pyridines, quinolines, nicotines, their derivatives and the like, which have anionic moieties attached thereto Most preferably, a sulphonate moiety is used
  • bathophenanthroline disulfonic acid BPDS
  • Additional chelating agents include phthalocyanine-tetrasulfonic acid, 2,2'-biquinoline-4,4'-disulfon ⁇ c acid, 4-hydroxy-7-sulfonyl-3-quinoline carboxylic acid, 2-hydroxy-5-sulfonyl pyridine-N-oxide, 2-hydroxy-5-sulfonyl-6-methylpyridine-3-carboxylic acid, ⁇ yridine-2-carboxyl-3-hydroxy-4-sulfonic acid and combinations thereof
  • One embodiment ofthe invention is directed to lanthanide chelates that reversibly bind targets that contain protonated amines
  • lanthanide chelates comprise at least one rare earth element such as a lanthanide and at least one ligand Suitable lanthanides include any one or more ofthe elements from the cerium or yttrium subgroups
  • the lanthanide is europium, terbium, samarium, gadolinium or dysprosium and a preferred embodiment comprises europium and bathophenanthroline disulfonic acid for reversible binding to targets such as proteins
  • Targets may be molecules such as amino acids, nucleotides, macromolecules such as proteins, peptides, polypeptides or nucleic acid, or modifications of these molecules or macromolecules Targets may be labeled while in solution or when immobilized on a solid (or semi-solid) supports such as nitrocellulose, PVDF, nylon or gels Because the procedure is fully reversible, labeled
  • lanthanide may be added first, last or simultaneously with the chelate Excess lanthanide chelate may be removed if desired Chelates are excited at a suitable wavelength of, for example, about 200 nm to about 400 nm, and preferably from about 280 nm to about 360 nm Phosphorescence is then measured at a wavelength appropriate for the lanthanide employed Emission wavelengths for each lanthanide are well known to those of ordinary skill or can be easily determined
  • one ofthe components is immobilized in some fashion on a solid (or semi-solid) support so that unreacted components may be washed away by washing the support
  • Target proteins for example, may be transferred to a solid support such as nitrocellulose membrane by electroblotting or capillary blotting.
  • Membrane is incubated with lanthanide chelate solubilized in some binding solution under conditions that favor protein-lanthanide chelate complex formation. Excess lanthanide chelate is removed by gently washing the membrane in binding solution that lacks lanthanide chelate.
  • the excess lanthanide chelate may be removed by dialysis, chromatography and like means.
  • Another method of removing excess reagents is to take advantage ofthe protein precipitating abilities of certain lanthanide chelates.
  • the lanthanide chelate will precipitate proteins at concentrations similar to those employed for labeling.
  • Complex formation may be performed in solution, the complex collected by centrifugation and phosphorescence measured either by first resuspending the pellet or by direct measurement of the phosphorescence of the pellet.
  • Lanthanide chelates may also be used in purification schemes.
  • targets such as proteins in a sample may be complexed with the lanthanide chelate and collected by centrifugation.
  • targets can be separated from other substances in the sample which do not luminesce. This can be done manually or automatically by, for example, fluorescence activated cell sorting (FACS) analysis or another automated procedure.
  • FACS fluorescence activated cell sorting
  • a simple assay measuring luminescence with increasing pH correlates the number of amine groups with pH stability. Binding stability is increased with an increased amino-to-carboxyl acid ratio. This fact can be used for a number of sophisticated applications.
  • a multi-detection immunoassay can be performed whereby one or more monoclonal antibodies are bound to one or more secondary antibodies as illustrated in Figure 1.
  • the secondary antibodies are tagged with poly-amines to increase the stability of binding to lanthanide chelates at a basic pH.
  • kits for the reversible detection of protein which contains a lanthanide and a chelate
  • the kit may further contain a binding solution and/or an elution solution
  • Kits may also contain various wash, elution or incubation solutions, as well as other buffers, stabilizing and storage solutions, and antibodies
  • Lanthanide chelates can be utilized as readily reversible labels for targets such as protein in solution or immobilized on a solid support Reversible target binding is best achieved by an anionic moiety such as sulfonate, sulfate, phosphonate or phosphate groups Binding may also be enhanced with aromatic or heteroaromatic functionalities
  • the procedure is simple and easy to perform, requiring reagents that are easily prepared in the laboratory, stored at room temperature for extended periods of time and can be reused several times without loss in labeling sensitivity
  • the labeling procedure is relatively inexpensive as it does not utilize precious metals such as gold
  • lanthanide chelate labeling requires 10-20 minutes to complete Quantitative stoichiometry of complex formation with proteins and peptides make lanthanide chelates also suitable for use in dot-blot assays for routine protein quantification Sensitivity is high, in the low nanogram range, and the dynamic range is quite broad, giving
  • lanthanide chelates do not modify targets such as proteins and nucleic acids, and are compatible with immunoblotting and protein microsequencing reagents and procedures
  • Lanthanide chelates do not cause N- or C-terminal blocking of proteins, interfere with the recognition of the tryptic cleavage sites, mask the antigenic recognition site for monoclonal anti-actin antibody or change the high pressure liquid chromatography (HPLC) or reverse phase (RP) HPLC elution profiles ofthe sequenced amino acids This makes the lanthanide chelates ideal for use in many applications where biochemical assays are to be performed subsequent to labeling
  • Chelates including 3-hydroxypicolinic acid and bathophenanthroline disulfonate (BPDS) (from Sigma Chemical Co , St Louis, MO), were evaluated for their ability to complex with lanthanides and enhance phosphorescence Chelates were combined with lanthanides and phosphorescence was measured on a spectrofluorometer Measurement can be optimized as described below, but generally for terbium complexes, time delay was set to 0 4 msec, while time gate was set to 4 1 msec For europium complexes, time delay was set to 0 05 msec, while time gate was set to 1 5 msec Terbium emission was monitored at 491 and 545 nm while europium emission is monitored at 590 nm and 615 nm Excitation wavelength should be in the 250 to 370 nm range
  • Membrane bound protein in a dot blot format was used to test the ability of lanthanide chelates to label protein Polyvinyl difluoride (PVDF) membrane (Immobilon-P, Oxford Glycosystems) or nitrocellulose membrane were dried and incubated in three changes of 50 mM sodium acetate, pH 4 0 for 5 minutes each while rocking on a rotary shaker (100 ⁇ ) Membranes were incubated in 0 1% polyvinyl pyrrolidone-40 in 50 mM sodium acetate, pH 4 0 for 5 minutes, if blocking was desired Membranes were incubated in 1 5 mM BPDS, 0 1 mM europium chloride (EuCl 3 , prepared in distilled water) for 10-15 minutes Membranes were rinsed twice in distilled water for 5 minute periods
  • EDTA and EGTA competed most efficiently for the lanthanide ion complexed to the protein (data not shown)
  • the lanthanide chelate can easily be eluted from the protein at a pH of 6 and above in the presence of 20 mM EDTA
  • the lanthanide chelate can also be eluted by incubating at basic pH without any EDTA.
  • MgCl 2 (20-100 mM) was relatively ineffective at eluting the colored complexes as was NaCl (20-100 mM), 10% acetic acid and 20% methanol.
  • Emission and excitation maxima may be determined by spectrofluorometry.
  • 6 mM BPDS and 0.5 mM EuCl 3 were used to generate solution spectra 1.9 ⁇ g/mm 2 BSA was applied to the membrane and labeled with the same solution.
  • Spectra were obtained using a Perkin-Elmer LS 50B spectrofluorometer with a quartz cuvette for the solution assay or a plate reader attachment for the solid-phase measurements.
  • Figure 2A shows the excitation spectra
  • Figure 2B shows emission spectra
  • the spectra for the lanthanide chelate (Eu/BPDS) in solution (solid lines) and bound to BSA (Eu/BPDS/BSA) immobilized on PVDF membrane (dotted lines) are shown
  • the data indicates that the excitation maxima for the trivalent Eu/PBDS/BSA complex lies between 285 nm and 300 nm and the emission maxima is between 605-615 nm
  • Example 5 Determination of Dynamic Range
  • Dynamic range is ascertained by measuring the phosphorescence of a variety of different concentrations of protein This was done by time-resolved phosphorescence detection of different concentrations of BSA using a Perkin-Elmer LS 50B spectrofluorometer.
  • BSA was applied to a PVDF membrane using a dot blot apparatus, the membrane dried and then labeled with 0 5 mM EuCl 3 and 6 mM BPDS.
  • Excitation wavelength was 293 nm, while emission wavelength was 615 nm
  • the time gate for measurement was 1 5 msec and the time delay was 0 05 msec Linear increase in signal with increasing concentration was obtained over a range of 15 to 476 ng (data not shown)
  • the data was replotted as a standard curve in Figure 3B
  • the X axis is the amount of protein per band divided by the square area ofthe band (ng/mm 2 )
  • the Y axis is the integrated intensity in arbitrary units (intensity of all the pixels in the image that belong to a particular band)
  • the Y-intercept was 1 5 and the slope was 0 43
  • the linear correlation coefficient ("r") ofthe best fit line through the data points was 0 99 From these two experiments, it was determined that the linear dynamic range was at least 1 9 ng/mm 2 to 476 ng/mm 2 (250-fold range)
  • Example 6 Optimization ofthe Lanthanide to Chelate Ratio Optimization of the ratio of europium to BPDS for detection of protein bound to nitrocellulose membrane was performed Europium concentration was maintained at 0 5 mM and BPDS concentration was varied The different compositions were evaluated for their ability to stain BSA (24 ng/mm 2 ) immobilized on nitrocellulose membrane The results are shown in
  • FIG. 5 shows the response curves for different proteins immobilized on nitrocellulose membrane and labeled with lanthanide chelate (0 1 mM Eu/ .5 mM BPDS)
  • the proteins evaluated are BSA (hollow circles), ovalbumin (filled diamonds), gelatin (filled triangles), phosvitin (crosses) and hemoglobin (filled circle)
  • Phosvitin is highly phosphorylated and the negative charge of the phosphate group is expected to inhibit interaction of the protein with the sulfonate moiety of BPDS
  • Hemoglobin is a highly colored protein and is likely to absorb emitted light from the europium Both of these proteins are refractory to lanthanide chelate labeling
  • the lanthanide chelate also binds to the amino acid glycine and the proteins urea and protamine sulfate (protamines are simple proteins that yield basic amino acids on hydrolysis and are found combined with nucleic acid in the sperm of fish) Primary amines seem to be required for lanthanide chelate labeling as TEMED does not generate a reaction
  • Acidic fractions (low pl) possess predominantly carboxyl moieties, while the basic fractions (high pi) contain mostly amino moieties.
  • ampholytes with a high amino to carboxyl ratio (high pl) are preferentially stained by the lanthanide chelate.
  • the acidic fractions labeled poorly Thus, the chelates appear to interact with amino groups and not carboxyl groups on proteins.
  • Example 1 1 Poly-amines are Resistant to Elution
  • the X axis is the polymer tested and the Y axis is the percent integrated intensity normalized to the intensity ofthe poly-allylamine signal
  • Poly-allylamine retained the complex to the greatest extent, followed by poly-L- lysine These are the polymers with the greatest number of primary amines (and thus the highest pl) and are therefore the most positively charged at pH 8 8
  • Example 12 Immunodetection of Proteins after Labeling
  • HL-60 human promyelocytic leukemia cells, ATCC CCL 240, American Type Culture Collection, Rockville, MD
  • protein extracts are prepared in 63 mM Tris, 2 0% sodium dodecyl sulfate, 10 0% glycerol, 5 0% 2-mercaptoethanol, pH 6 8 and heated to 100°C for 10 minutes
  • Va ⁇ ous amounts ofthe cell lysate (0 5 ⁇ g to 10 0 ⁇ g) are run on 10 0% polyacrylamide gels by standard protocols
  • label is eluted from the membrane as described
  • the nitrocellulose membranes are blocked in 5 0% nonfat dry milk in PBS and immunodetection performed using the standard ECL Western blotting protocol (Amersham Interna ⁇ tional, Buckinghamshire, England), utilizing monoclonal anti-actin antibody, clone KJ43A (S)

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  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
EP96941407A 1995-11-30 1996-11-19 Lumineszierende sonden zum nachweis von proteinen Withdrawn EP0807254A1 (de)

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US56495395A 1995-11-30 1995-11-30
US564953 1995-11-30
PCT/US1996/018575 WO1997020213A1 (en) 1995-11-30 1996-11-19 Luminescent probes for protein detection

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ATE349700T1 (de) 1998-10-27 2007-01-15 Molecular Probes Inc Übergangsmetallkomplex-haltige lumineszente proteinfarben
US6329205B1 (en) 1999-08-31 2001-12-11 Molecular Probes, Inc. Detection method using luminescent europium-based protein stains
AU2003285969A1 (en) * 2002-11-25 2004-06-18 Albert Missbichler Method for detecting membrane-bound and/or amyloid proteins
US9193746B2 (en) 2006-12-07 2015-11-24 Biotium, Inc. Luminescent metal complexes and associated technology
FR2943788B1 (fr) 2009-03-24 2016-12-30 Hopitaux Paris Assist Publique Procede de prediction d'un reflux vesico ureteral de haut grade chez les enfants avec une premiere infection urinaire febrile
CN103293300A (zh) * 2012-07-09 2013-09-11 深圳市艾瑞生物科技有限公司 基于磷光发光技术的时间分辨荧光检测试剂盒及其制备方法和应用
FR3008186B1 (fr) * 2013-07-04 2016-12-09 Commissariat Energie Atomique Procede de detection non destructif d'interactions proteines uranium sur gels d'electrophorese
US12405185B2 (en) 2022-02-28 2025-09-02 David Putnam Leak-and-sealing-failure-detection sensor and system

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EP0171978B1 (de) * 1984-08-13 1990-11-07 HSC Research Development Corporation 1,10-Phenanthrolin-2,9-dicarbonsäure-Derivate und deren Verwendung für Fluoreszens-Immunoassay
US5071775A (en) * 1986-08-13 1991-12-10 Massachusetts Institute Of Technology Indirect labeling method for post-separation detection of chemical compounds
GB8927503D0 (en) * 1989-12-04 1990-02-07 Kronem Systems Inc Enzyme-amplified lanthanide chelate luminescence

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AU1055997A (en) 1997-06-19

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