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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • 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
  • Y10S435/975—Kit
• • 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
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/815—Test for named compound or class of compounds

Definitions

• the invention relates to a new homogeneous method for the detection and / or determination of the phosphorylation activity (or phosphorylating activity) of a biological material with respect to a substrate containing tyrosine and or serine and / or threonine, as well as a kit for the implementation of this method.
• the phosphorylation of biological molecules such as peptides or proteins by kinases is a major biological mechanism for regulating cell metabolism.
• the detection of phosphorylation of the substrate fixed on a solid phase is done either by measuring the incorporation of 3 p in the enzyme substrate, or by the use of a labeled antibody (isotopic tracer, enzymatic or fluorescent) directed against the phosphorylation site.
• a labeled antibody isotopic tracer, enzymatic or fluorescent
• the amount of phosphorylated substrate is revealed by measuring the signal emitted by the luminescent carrier molecule and generated by energy transfer from a specific receptor for the phosphorylated substrate labeled by a luminescent molecule.
• This method is particularly useful for measuring the phosphorylation of molecules of biological interest, such as for example peptides, polypeptides, proteins or nucleotides, in natural or pathological processes, or during synthetic processes such as for example synthesis nucleic acids or proteins.
• the invention therefore relates to a homogeneous method for the detection and / or determination of the phosphorylating activity of a biological material with respect to a substrate containing tyrosine and / or serine and / or threonine, characterized in that said biological material is brought into contact with a plurality of peptides or polypeptides containing the same or different tyrosine and / or serine and / or threonine linked covalently to a carrier molecule, in the presence of a source of non-radiolabelled phosphate and of receptors specific for said phosphorylated peptides or polypeptides, and in that the detection and / or determination of phosphorylating activity is carried out by measuring a transmission signal, said emission signal resulting from an interaction between said carrier molecule constituted by a luminescent molecule or a non-luminescent molecule linked to at least one luminescent marker or a modulator of the emission signal and said specific receptors linked to at least one
• luminescent marker is meant a luminescent molecule used to detect the interaction between the carrier molecule and the specific receptor.
• emission signal modulator is meant a molecule which, when present near a luminescent molecule, modifies the characteristics of the emission signal thereof.
• the same luminescent compound can play the role of luminescent marker or modulator of the emission signal.
• Said modulator may be a luminescent molecule, for example a donor or acceptor luminescent molecule, or a non-luminescent molecule, for example an atom with a high atomic number or a molecule containing such an atom as described for example in application EP 0 232 348, or even spin marker compounds.
• a luminescent molecule for example a donor or acceptor luminescent molecule
• a non-luminescent molecule for example an atom with a high atomic number or a molecule containing such an atom as described for example in application EP 0 232 348, or even spin marker compounds.
• the carrier molecule can be:
• the carrier molecule can therefore be either an acceptor luminescent molecule or a non-luminescent molecule linked to at least one luminescent marker or to at least one modulator of the emission signal.
• molecule or “compound” will be used without distinction to qualify the luminescent markers or the modulators linked to the carrier molecule or to the specific receptor.
• the carrier molecule is either a fluorescent acceptor molecule, or a fluorescent donor molecule, or a non-fluorescent molecule linked to at least one fluorescent acceptor compound, or to at least one fluorescent donor compound.
• the luminescent marker or the modulator of the emission signal linked to each of the specific receptors of the phosphorylated peptide (s) or polypeptide (s) can be a fluorescent donor or acceptor molecule.
• the detection and / or the determination of the phosphorylation activity is carried out by measuring the emission signal resulting from the transfer of non-radiative energy between the carrier molecule and the luminescent markers or the modulators of the signal. emission linked to specific receptors for phosphorylated peptides or polypeptides.
• the light emission signal allowing the detection and / or determination of the desired phosphorylating activity can be generated either by transfer of non-radiative energy from the luminescent markers or modulators of the emission signal linked to the specific receptors for the carrier molecule, either inversely by transfer of non-radiative energy from the luminescent markers or modulators of the emission signal of the carrier molecule to the luminescent markers linked to the specific receptors.
• the acceptor compound has an absorption spectrum which covers at least partially the emission spectrum of the donor and has a high molar absorbance in this overlap region, and an emission spectrum in a range of length waves where the donor has a weak intrinsic emission;
• the acceptor and the donor are located close to each other.
• the amount of peptides or polypeptides covalently linked to the carrier luminescent molecule can be from about 2 to 1000 per carrier luminescent molecule.
• the specific receptors for phosphorylated peptides or polypeptides can, for example, be chosen from monoclonal or polyclonal antibodies.
• the luminescent compound linked to the specific receptor of the phosphorylated peptide (s) or polypeptide (s) or to the carrier molecule, as luminescent marker or modulator of the emission signal according to the mechanism of the interaction between said carrier molecule and said specific receptor is a chelate, a cryptate or a macrocyclic complex of rare earth ion.
• Said donor fluorescent compound is preferably a rare earth cryptate preferably chosen from terbium, europium, samarium or dysprosium cryptates.
• said rare earth cryptate consists of at least one rare earth salt complexed with a macropolycyclic compound of formula
• Z is an atom having 3 or 4 valences
• R is nothing or represents hydrogen
• the bivalent radicals ⁇ , OJ) and ⁇ are independently one of l other hydrocarbon chains which optionally contain one or more heteroatoms and are optionally interrupted by a heteromacrocycle, at least one of the radicals ®, () and ⁇ additionally comprising at least one molecular unit or essentially consisting of a molecular unit, said molecular motif having a triplet energy higher than that of the emissive level of the complexed rare earth ion.
• the molecular unit is chosen from phenanthroline, anthracene, benzene, naphthalene, bi- and ter-phenyl, azobenzene, azopyridine, pyridine, bipyridines, bisquinolines and the compounds of the following formulas:
• Xi and X 2 which may be identical or different denote oxygen, nitrogen or sulfur,
• X being oxygen or hydrogen.
• the fluorescent compound is a rare earth cryptate consisting of the terbium or europium ion complexed by one of the macrocyclic compounds below:
• a particularly advantageous fluorescent compound is the europium cryptate Eu tris bipyridine.
• macrocyclic compounds complexing rare earth ions in which the molecular unit is chosen from bipyrazines, bipyrimidines and nitrogen heterocydes containing N-oxide groups.
• Macrocyte compounds with bipyrazine units are described in F. Bodar-Houillon et al., New J. Chem., 1996, 20, 1041-1045. Macrocyclic compounds with bipyrimidine units are described in JM Lehn et al., Helv. Chim. Acta, 1992, 75, 1221.
• Macrocyclic compounds comprising nitrogen heterocydes comprising N-oxide groups are described in J.M. Lehn et al., Helv. Chim. Acta, 1991, 74, 572 and in patent EP 0 601 113.
• the rare earth cryptate used as the fluorescent donor compound can also consist of at least one rare earth salt complexed with a macropolycyciic compound corresponding to one of the formulas II or III below:
• - Y is a group or a spacer arm which is constituted by a bivalent organic radical, chosen from linear or branched alkylene groups in d to C 20 optionally containing one or more double bonds or triple bonds and / or possibly being interrupted by one or more heteroatoms such as oxygen, nitrogen, sulfur or phosphorus, from C 5 to C 6 cycloalkylene groups or from C 1 to C 14 arylene groups, said alkylene, cycloalkylene or arylene groups being optionally substituted with alkyl, aryl or sulfonate groups;
• - Z is a functional group capable of binding covalently with a biological substance
• - R is a methyl group or represents the group -YZ; - R 'is hydrogen or a group -COOR "in which R" is a C 1 to C 10 alkyl group and preferably represents the methyl, ethyl or tert-butyl group or else R' is a group -CO-NH- YZ.
• said fluorescent compound can be linked to the specific receptor or to the carrier molecule either directly or via an arm of spacing.
• This spacer arm for example consists of a divalent organic radical selected from linear or branched alkylene groups, C 1 -C 2 0, optionally containing one or more double bonds and / or optionally interrupted by one or more heteroatoms, such than oxygen, nitrogen, sulfur or phosphorus; carbamoyl and carboxamido groups; C 5 -C 8 cycloalkylene groups and C ⁇ -C ⁇ 4 arylene groups, said alkylene, cycloalkylene or arylene groups being optionally substituted by alkyl, aryl or sulfonate groups.
• a divalent organic radical selected from linear or branched alkylene groups, C 1 -C 2 0, optionally containing one or more double bonds and / or optionally interrupted by one or more heteroatoms, such than oxygen, nitrogen, sulfur or phosphorus; carbamoyl and carboxamido groups; C 5 -C 8 cycloalkylene groups and C ⁇ -C ⁇ 4 arylene groups, said alkylene, cycl
• a fluorescent donor compound linked to the specific receptor of a europium cryptate and, as a carrier molecule or fluorescent acceptor compound linked to the carrier molecule, allophycocyanin, allophycocyanin B, a derivative of chemically modified allophycocyanin, C phycocyanin, R phycocyanin and cyanines.
• a terbium cryptate will be used as the donor fluorescent compound linked to the specific receptor and as a carrier molecule or fluorescent acceptor compound linked to the carrier molecule, rhodamines, thionine, R phycocyanin, phycoerythrocyanin , C phycoerythrine, B phycoerythrine, R phycoerythrine and cyanines.
• Fluorescent compounds which can also be used as acceptor compounds are the phycobiliprotein-binding peptide complexes described in application WO96 / 42016.
• the invention also relates to a kit for the detection and / or determination of the phosphorylating activity of a biological material with respect to a substrate containing and and / or serine and / or from threonine, characterized in that it contains at least one carrier molecule to which are covalently attached a plurality of identical or different peptides or polypeptides, and at least one receptor specific for said phosphorylated peptides or polypeptides, said receptor being linked to at least one luminescent marker or a modulator of the emission signal.
• the carrier molecule is as defined above, that is to say that it can be luminescent intrinsically or by binding to at least one luminescent marker or a modulator of the emission signal.
• the carrier molecule and the luminescent marker or the modulator of the emission signal linked to the specific receptor of this kit are fluorescent compounds.
• the luminescent compound (luminescent marker or modulator of the emission signal) linked to the specific receptor and the carrier molecule are respectively fluorescent donor and acceptor compounds.
• the luminescent compound linked to the specific receptor in the kit according to the invention may be the europium cryptate Eu trisbipyridine or the terbium cryptate Tb trisbipyridine.
• the kit according to the invention also contains an appropriate buffer medium, a source of non-radiolabelled phosphate and instructions for the implementation of the method of detection and / or of the determination of the phosphorylating activity of a material. biological described above.
• the SRC peptide is a substrate of the epidermal growth factor receptor tyrosine kinase, also known as EGF for "Epidermal Growth Factor". It is a peptide of 11 amino acids containing a single tyrosine motif and having the following structure:
• EuTBP europium cryptate Eu trisbipyridine diamine
• BSA bovine serum albumin
• IgG immunoglobulin G
• MHS maleimidohexanoyl-N-hydroxy-succinimidester
• Sulfo-SMCC sulfosuccinimidyl 4-n-maleimidomethyl) cyclohexane.
• a chemically modified allophycocyanin derivative (XU ⁇ s, Cis bio international) is used, the high molecular weight of which allows it to be labeled with numerous peptides each having a phosphorylation site.
• the uncoupled peptide is removed by exclusion-diffusion chromatography on a TSK 3000 SW column (MERCK) in phosphate buffer 100 mM pH 7.0.
• a conjugate comprising between 20 and 40 peptides per molecule
• IgG PY20 Activation of IgG PY20 by SPDP 5 mg of IgG PY20 (Transduction Laboratories) at a rate of 10 mg / ml in 100 mM phosphate buffer, pH 7.0 are activated by the addition of a solution of SPDP ( Pierce, USA) at 6.4 mM in dioxane in a molar ratio of 7.5 SPDP by IgG P420.
• the pyridine-2-thione IgG is purified on a G25 superfine column in a 100 mM phosphate buffer, 5 mM EDTA, pH 6.5.
• the proteins are concentrated and the 2-pyridyl disulfide groups are reduced by a solution of DTT (Sigma, USA) having a final concentration of
• reaction medium After 45 min of activation at room temperature, the reaction medium is filtered at 0.8 ⁇ m in order to remove the precipitate which may have formed.
• Undesirable reaction products sulfo-SMCC, N-hydroxysuccinimide, (N-maleimidomethyl) carboxylic acid
• sulfo-SMCC N-hydroxysuccinimide, (N-maleimidomethyl) carboxylic acid
• ion exchange chromatography on a Mono Q column (Pharmacia, Sweden) in 20 mM phosphate buffer dimethylformamide 10% (v / v ), pH 7.0 under NaCl shock.
• concentration of Eu TBP maleimide is determined at 307 nm with an 8307 nm
• the maleimide functions are reacted with the thiol functions attached to the antibody, in molar proportions varying from 10 to 30 Eu TBP maleimide with IgG PY20-SH. After 18 hours of incubation at 4 ° C and blocking of thiol groups
• the uncoupled Eu TBP is removed by dialysis in 100 Mm phosphate buffer pH 7.0 at 4 ° C until exhaustion (more fluorescence in the dialysis baths).
• the characteristics of the conjugate are determined by its absorptions at 307 nm and at 280 nm using the following values taking into account the inherent absorption of the cryptate determined by the ratio A307nm / A280nm- Eu TBP-maleimide:
• A431 cells (SIGMA) containing an EGF receptor are preactivated for 10 min at room temperature with EGF.
• the phosphorylation buffer is a 60 mM TRIS / MES buffer, pH 7.4 containing 30 ⁇ M ATP, 50 mM Mg ++ and 10 mM Mn ++ .
• a nitrogen pulsed laser (LASER SCIENCE INC., Model LS1-337ND) is used as an excitation source (wavelength at 337J nm). Pulse duration is specified at 3 nanoseconds and is repeated at a frequency of 10 Hertz.
• the beam passes through a filter (CORNING) in order to eliminate any stray light at excitation other than 337 nm.
• the beam After entering the measurement chamber, the beam is reflected by a dichroic filter, placed at 45 degrees, which has the property of reflecting ultraviolet light and of being able to transmit visible light.
• the beam reflected by the dichroic filter is focused on the well to be measured of a microplate by a lens of fused silica.
• the fluorescence emission is collected at a solid angle of 20 degrees, collimated by the same lens, and passes directly through the dichroic filter (fluorescence in visible light).
• An interference filter of characteristics defined according to the fluorescence wavelength to be detected makes it possible to get rid of the lights which can parasitize the signal, the intensity of which is then measured by a photomultiplier (HAMAMATSU R2949).
• the photon counter used is an SR-400 (STANFORD RESEARCH
• SYSTEMS whose operations and synchronization with the laser are controlled by an IBM PC-AT type computer via an RS 232 output.
• the pulses from the photomultiplier are recorded during a time window (tg) and after a delay (t ⁇ ) determined on condition that they are greater than a discriminating level selected by the photon counter in order to optimize the signal / noise ratio of the photomultiplier.
• An XY table controlled by the IBM PC-AT, allows the different positioning of the measurement microplate by stepping motors, including the loading, positioning under the exciting beam, automatic sequential reading of the 96 wells , and exit.
• the fluorescence emitted by the XL665-SRC peptide conjugate is measured using the prototype fluorimeter equipped with a 665 nm filter 10 nm wide at half height, for 400 ⁇ s and with a delay of 50 ⁇ s.
• the concentration of Xl_665-SRC peptide conjugate is expressed in nM of XL-665-

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• 1997
  • 1997-09-19 FR FR9711721A patent/FR2768817B1/fr not_active Expired - Fee Related
• 1998
  • 1998-09-16 EP EP98943995A patent/EP1018009B1/fr not_active Expired - Lifetime
  • 1998-09-16 DE DE69813850T patent/DE69813850T2/de not_active Expired - Fee Related
  • 1998-09-16 JP JP2000513143A patent/JP4312953B2/ja not_active Expired - Fee Related
  • 1998-09-16 ES ES98943995T patent/ES2198744T3/es not_active Expired - Lifetime
  • 1998-09-16 AT AT98943995T patent/ATE238551T1/de not_active IP Right Cessation
  • 1998-09-16 WO PCT/FR1998/001976 patent/WO1999015896A1/fr not_active Ceased
  • 1998-09-16 US US09/486,701 patent/US6753156B1/en not_active Expired - Fee Related

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