JPH1048217A - Method and kit for measuring activity of enzyme for phosphating protein kinase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure the activity of enzyme for phosphating various protein kinase easily with high sensitivity. SOLUTION: The inventive method comprises step (a) for causing a labeled substrate peptide to be phosphated by protein kinase to react on the protein kinase in liquid phase to produce phosphated labeled substrate peptide, step (b) for causing an antibody, which is boded specifically with the phosphated labeled substrate peptide (where specified amino acid in the amino acid sequence of substrate peptide is phosphated), to be bonded with an insoluble support thus producing a solid phase antibody, and step (c) for causing the solid phase antibody to react on the phosphated labeled substrate peptide produced in step (a) thus bonding them. Subsequently, the labeled quantity is measured for the bonded product of the solid phase antibody produced in step (b) and the phosphated labeled substrate peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method and a kit for measuring the kinase activity of protein kinase.

[0002]

BACKGROUND OF THE INVENTION Regardless of whether it is a multicellular organism or a unicellular organism,
All cells respond by taking in the necessary information from outside. In a multicellular organism, since each cell takes in appropriate information and responds appropriately, an extremely complicated individual is created through the process of development and its homeostasis is maintained. Information from outside the cell is exchanged through humoral factors secreted by other cells or through contact between cells, but this information is sensed by receptors on the cell, and the signal is transmitted inside the cell. It traverses a complex pathway and triggers various responses with gene expression. The onset of proliferation and differentiation is the most striking cellular response. The process by which extracellular information is transmitted through the cell membrane into the cell and then transmitted to the cell nucleus is called intracellular information transmission, and the process involves the phosphorylation cascade of many proteins. Are known.

[0003] Phosphorylation of intracellular proteins is carried out by a protein kinase (protein kinase). Examples of such protein kinases include protein kinase C (PKC), cyclic AMP-dependent protein kinase (PKA), and PI3. Kinases, cdc2 kinases, raf-1 kinases and the like are known, and these protein kinases are involved in the regulation of cell growth and differentiation through intracellular signaling, and are involved in protein kinases and cell signaling. Abnormal expression of a molecule is expressed as a disease such as cancer or diabetes. Therefore, detection of dysfunction of these protein kinases and molecules involved in intracellular signal transduction is indispensable for elucidation of diseases such as cell proliferation and cell differentiation and cancer. As a means for detecting protein kinase dysfunction, the enzyme activity of protein kinase is measured, and a radioimmunoassay (RI
A) and enzyme-linked immunosorbent assay (ELISA) were known.

[0004]

However, among these conventional methods, the RIA method requires a facility for handling radioactive materials, so that it has been difficult to perform a simple measurement. In the ELISA method, a substrate peptide to be phosphorylated is bound to a solid phase, the substrate peptide is phosphorylated by a protein kinase in a sample, and then a labeled antibody (an antibody obtained by labeling an antibody against the phosphorylated substrate peptide). ), And the degree of kinase activity was detected by detecting the amount of labeling of the reaction product. However, in this method, since the amount of substrate peptide bound to the solid phase is limited, protein kinase The reaction efficiency was poor, and phosphorylation was sometimes not performed sufficiently. That is, as a measurement object, those having a relatively large Km value (Michaelis constant) such as PKC and PKA and requiring no high sensitivity can sufficiently measure phosphorylation enzyme activity because phosphorylation proceeds sufficiently even by such a method. However, in the case of those having a small Km value, such as cdc2 kinase and raf-1 kinase, phosphorylation does not proceed sufficiently by the method using a phosphorylated substrate peptide bound to a solid phase, and it is impossible to measure with high sensitivity. It was possible.

For this reason, not only protein kinases having a large Km value but also protein kinases such as cdc2 kinase having a not so large Km value can be used to measure the kinase activity, that is, various methods can be used. It has been desired to establish a method for measuring the kinase activity of protein kinase.

The present invention has been made in view of the above problems, and provides a measuring method and a measuring kit capable of measuring the kinase activity of various protein kinases with high sensitivity and ease.

[0007]

Means for Solving the Problems The first method of the present invention for measuring the kinase activity of protein kinase is characterized by comprising the following steps (a) to (c). That is, (a) reacting, in a liquid phase, a labeled substrate peptide obtained by labeling a substrate peptide that can be phosphorylated by a protein kinase with a labeling substance, and the protein kinase;
A step of preparing a phosphorylated label substrate peptide. (B) an immobilized antibody obtained by binding an antibody that specifically binds to a phosphorylated substrate peptide (a predetermined amino acid in the amino acid sequence of the substrate peptide is phosphorylated) to an insoluble support; A) reacting the phosphorylated label substrate peptide obtained in the step 2) to bind them. (C) a step of measuring a labeling amount of a conjugate of the immobilized antibody obtained in the above (b) and the phosphorylated label substrate peptide.

A kit for measuring protein kinase phosphorylase activity, which is the second of the present invention, comprises a labeled substrate peptide obtained by labeling a substrate peptide that can be phosphorylated by protein kinase with a labeling substance, and a phosphorylated substrate peptide (substrate peptide). And a solid-phased antibody obtained by binding an antibody that specifically binds to a specific amino acid in the amino acid sequence of (1) to an insoluble support.

According to the present invention, the Km value, which has been difficult to measure the kinase activity, is not so large.
Particularly useful for dc2 kinase and the like. That is, the protein kinase is cdc2 kinase and the substrate peptide is a synthetic peptide of mouse vimentin M
V55 peptide (peptide represented by SEQ ID NO: 1 in the sequence listing), wherein the phosphorylated substrate peptide is phosphorylated MV
55 peptide (the serine at amino acid number 6 of the peptide represented by SEQ ID NO: 1 in the sequence listing is phosphorylated), and the antibody is an anti-phosphorylated MV55 peptide antibody (for example, fusion cell clone 4A4 (accession number: FERMP
Monoclonal antibody produced by -15759)
Even in this case, the kinase activity can be measured with high sensitivity.

[0010] As the labeling substance, for example, one selected from the group consisting of biotin, peroxidase, β-D-galactosidase, alkaline phosphatase and microperoxidase can be used.
The measurement kit includes, in addition to the labeled substrate peptide and the immobilized antibody, for example, a substrate for measuring the labeling amount of the labeling substance, a coloring agent, and the like, and a protein kinase standard used for obtaining a calibration curve. It may be provided with a substance (purified product).

[0011]

DETAILED DESCRIPTION OF THE INVENTION As in the prior art, a substrate peptide is bound to an insoluble support, and this is reacted with a protein kinase to form a phosphorylated substrate peptide, which is labeled to specifically bind to the phosphorylated substrate peptide. In a measurement system in which an antibody is reacted and the amount of the label is measured, the reaction efficiency is not good because phosphorylation is a solid-liquid reaction. For this reason, this assay has a relatively large Km value and does not require high sensitivity (for example, PKC and PKC).
A), but can be applied to cdc2 kinase or r
It was difficult to apply to a protein such as af-1 kinase, whose Km value was not so large and the substrate peptide could not be phosphorylated efficiently. This time, Km value is not so large because phosphorylation of substrate peptide is performed in liquid phase.
Even with dc2 kinase or the like, phosphorylation proceeded efficiently, and highly sensitive measurement of kinase activity was realized.

[Regarding Substrate Peptide] The kinase activity of protein kinase is measured by detecting phosphorylated substrate peptide. Examples of the substrate peptide to be phosphorylated by protein kinase include MV55 peptide (peptide of SEQ ID NO: 1 in the sequence listing, in which case serine of amino acid number 6 is phosphorylated) when the protein kinase is cdc2 kinase, r
In the case of af-1 kinase, the MEK-1 peptide (the peptide of SEQ ID NO: 2 in the sequence listing, in which case serine at amino acids 6 and 10 is phosphorylated), PKC or PK
In the case of KA, it is preferable to use a PS peptide (the peptide of SEQ ID NO: 3 in the sequence listing, in which case serine at amino acid number 7 is phosphorylated). Such a substrate peptide can be synthesized, for example, using a fully automatic synthesizer.

[Regarding Labeling Substance] Labeling substances include:
It can be used without any particular limitation as long as it can be used for ordinary immunological measurement methods, for example, enzymes, biotin,
A fluorescent substance, a luminescent substance, a radioactive substance, and the like can be used. Among them, an enzyme and biotin are preferable from the viewpoint of easy measurement. Enzymes include peroxidase, β
-D-galactosidase, alkaline phosphatase,
Microperoxidase. Examples of the fluorescent substance, fluorescein isothiocyanate, phycobiliprotein, phycoerythrin and the like, and the luminescent substance Isorushinoru, lucigenin and the like, and as the radioactive material ¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H and the like.
Further, when biotin is used as the labeling substance, the amount of labeled biotin can be measured with high sensitivity by further using enzyme-labeled avidin, which is preferable. As the enzyme of the enzyme-labeled avidin, the same enzyme as that used when the antibody is labeled can be used, but peroxidase is preferable.

[About anti-phosphorylated substrate peptide antibody]
Antibodies that specifically bind to phosphorylated substrate peptides are described in the following A.I. ~ F. Can be obtained as follows. A. Antigen As the antigen, a phosphorylated substrate peptide (for example, phosphorylated MV when the protein kinase is cdc2 kinase)
55 peptides (the serine at amino acid No. 6 of the peptide represented by SEQ ID NO: 1 is phosphorylated), and phosphorylated ME when the protein kinase is raf-1 kinase
K-1 peptide (the serine at amino acids 6 and 10 of the peptide represented by SEQ ID NO: 2 is phosphorylated),
When the protein kinase is PKC or PKA, a phosphorylated PS peptide (the serine at amino acid number 7 of the peptide represented by SEQ ID NO: 3 is phosphorylated) is synthesized and used as an immunogen. Since the peptide is a relatively small molecule and it is difficult to produce an antibody even when the peptide is immunized as it is, KLH (keyhole limpet hemocyanin) is usually used.
It is preferable to use it as an immunogen by binding it to a carrier protein such as albumin.

B. Immunization with the above antigens As immunized animals, besides mice which are mammals, rats,
Hamsters and the like can also be used. Usually, mice are the most widely used, and BALB / c mice and other strains (str
ain) mice can be used. In this case, the immunization scheme and the concentration of the antigen should be selected so that a sufficient amount of antigen-stimulated lymphocytes is formed. For example, one mouse is immunized three times into the peritoneal cavity at intervals of two weeks with 25 μg of the antigen, and then 25 μg of the antigen is intravenously administered. Several days after the final immunization, spleen cells are removed for fusion.

C. Cell fusion The spleen is aseptically removed from the mammal individual immunized as described above, and a single cell suspension is prepared therefrom. The spleen cells (antibody-producing cells) are fused with a suitable myeloma cell by using a suitable fusion promoter. The myeloma cells are preferably derived from mammals of the same species as the immunized animal, but spleen cells of rats, hamsters and the like can be fused with mouse myeloma cells. The preferred ratio of spleen cells to myeloma cells ranges from about 20: 1 to about 2: 1. Is appropriate use of a fusion medium 0.5~1.5ml for about 10 ⁸ splenocytes. As a preferred fusion promoter, for example, polyethylene glycol having an average molecular weight of 1,000 to 4,000 can be advantageously used, but other fusion promoters known in the art (for example, Sendai virus (also known as HVJ)) can also be used. Cell fusion may be performed by a method using electric shock other than the method using these fusion promoters.

D. Selection of fused cells producing the desired monoclonal antibody In a separate container (eg, a microtiter plate), a mixture of unfused splenocytes, unfused myeloma cells, and fused fusion cells is used to support unfused myeloma cells. Diluted in selective medium without, and for a time sufficient to kill unfused cells (approximately 1
Time) Incubate. As the medium, a medium that is drug-resistant (for example, 8-azaguanine-resistant) and does not support unfused myeloma cells (for example, HAT medium (medium containing hypoxanthine, aminopterin, and thymidine)) is used. Unfused myeloma cells die in this selective medium. Unfused splenocytes are non-neoplastic cells, and die after a certain period of time (eg, one week). In contrast, the fused cells
Since it has the properties of parental spleen cells and the tumor properties of parental cells of myeloma, it can survive in a selective medium.

After the detection of the fused cells, an antibody against the phosphorylated substrate peptide is screened by an enzyme-linked immunosorbent assay (Enzyme Linked Immunosorbent Assay), and a monoclonal antibody that specifically binds to the phosphorylated substrate peptide is identified. Only the fusion cells to be produced are selected. For example, as a fusion cell producing an anti-phosphorylated MV55 peptide antibody, clone 4A4 (accession number:
FERM P-15759) and clone 1E9 as a fusion cell producing an anti-phosphorylated MEK-1 peptide antibody.
(Accession number: FERM P-15993) and clone 2F11 (Accession number: FERM P-15994), and clone 2B9 (Accession number: FERM P-1513) as a fusion cell producing an anti-phosphorylated PS peptide antibody
3) and clone 2G3 (Accession number: FERM P-1)
5134).

F. Obtaining the desired monoclonal antibody After cloning the fused cells producing the desired monoclonal antibody by an appropriate method (eg, limiting dilution method), the antibody can be produced by two different methods. According to the first method, a monoclonal antibody produced by the fusion cell can be obtained from the culture supernatant by culturing the fusion cell for a certain period in an appropriate medium. According to a second method, the fused cells can be injected into the peritoneal cavity of an immunized animal having an isogenic or semi-isogenic gene. From the blood and ascites of the host animal after a certain time,
A monoclonal antibody produced by the fusion cell can be obtained.

[Immobilized antibody] An immobilized antibody is obtained by binding an anti-phosphorylation substrate peptide antibody to an insoluble support. Such an immobilized antibody can be obtained, for example, by contacting a solution of the antibody with an insoluble support to adsorb the antibody to the surface of the insoluble support, or by binding the antibody by a chemical method such as a covalent bond. You can also get. The anti-phosphorylation substrate peptide antibody may be a complete antibody or a fragment such as Fab or F (ab) ' ₂ .

Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran,
Examples include polymers such as polysaccharides, paper, glass, metals, agarose, and combinations thereof. In addition, the shape of the insoluble carrier may be various shapes such as a tray, a sphere, a bar, a fiber, a disk, a container, a cell, and a test tube.

[Measurement] When an enzyme is used as a labeling substance, a substrate and, if necessary, a color former are used to measure its activity. When peroxidase is used as enzyme, using H ₂ O ₂ as substrate, as a color-developing agent 2,2'-azino - di - [3 ethylbenzthiazoline sulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, o-phenylenediamine (OPD), 4
-Aminoantipyrine, 3,3 ', 5,5'-tetramethylbenzidine and the like. When alkaline phosphatase is used as an enzyme, o-nitrophenyl phosphate is used as a substrate. When β-D-galactosidase is used as an enzyme, as a substrate. Fluorescein-di- (β-D-galactopyranoside), 4-methylumbelliferyl-β-D-
Galactopyranoside and the like can be used.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below.
The embodiments of the present invention are not limited to the following examples at all, and it goes without saying that various embodiments can be adopted as long as they belong to the technical scope of the present invention.

Example 1 Preparation of Immunogen Phosphorylated MV55 peptide (having serine at amino acid number 6 of the peptide represented by SEQ ID NO: 1 in the sequence listing) was synthesized by Millipore 9050 fully automatic synthesizer. Was prepared by a solid phase method using the Fmoc method in accordance with the instruction manual of the apparatus (for details of the synthesis method, see Shujunsha, Cell Engineering Supplement, “Anti-Peptide Antibody Experiment Protocol”). A cysteine residue for binding to a carrier protein was introduced at the C-terminus. The synthesized phosphorylated peptide was cleaved and the protecting group was removed with trifluoroacetic acid, and then purified by reversed-phase high-performance liquid chromatography under the following conditions. Column used: μBondasphere 5-μm C18 100A (3.9 × 1
Elution conditions: 0.1% trinitroacetic acid (acetonitrile 6)
~ 60% linear gradient)

The phosphorylated MV55 peptide was bound to keyhole limpet hemocyanin (KLH) by the MBS method to obtain an immunogen. That is, 4 mg of KLH is added to 0.25 ml of 10 m
M-phosphate buffer (pH 7.2), and 0.7 mg of 3-maleimidobenzoic acid-acid dissolved in 20 μl of dimethylformamide (DMF).
N-hydroxysuccinimide ester (MBS) was added and reacted at room temperature for 30 minutes. After the reaction, gel filtration was performed using a column (1.5 × 10 cm) packed with Sephadex G25, and the KLH-MB complex and free KLH
Was separated and the KLH-MB complex was collected. Then, 5 mg of the phosphorylated MV55 peptide was dissolved in 1 ml of a 0.1 M borate buffer (pH 9.0), and this solution and KLH-
The MB complex solution was mixed, the pH was adjusted to 7.0 to 7.5, and left at room temperature for 3 hours to react. After the reaction, PBS
After sufficient dialysis against (physiological phosphate buffer), the mixture was adjusted to a concentration of 0.5 mg / ml with PBS, dispensed in 100 μl aliquots, and frozen at −20 ° C. for storage.

[Example 2] Immunization of mice 100 µl of the immunogen (phosphorylated MV55 peptide-KLH) cryopreserved in Example 1 and 100 µl of complete Freund's adjuvant were mixed well to prepare a suspension. Solution was injected intraperitoneally into BDF1 mice as antigen
Each 5 μg was administered. Two weeks later, the same amount of antigen was administered again, and three days later, the spleen was removed, and cell fusion was performed as described below.

Example 3 Cell Fusion and Selection and Acquisition of Fusion Cells Producing the Desired Monoclonal Antibody [0027] The spleen cells of the excised mouse and myeloma cells (SP2 / 0-Ag14) of the syngeneic mouse were subjected to approx. , And cell fusion was performed using 50% polyethylene glycol 4000 as a fusion promoter. 1 × 10 ⁶ cells after fusion
The cells were suspended in a HAT medium containing 10% bovine serum (a medium containing hypoxanthine, aminopterin, and thymidine) so as to have a cell concentration of cells / ml, and 96-well microtiter plates (Maxi Soap manufactured by Nunc Corporation, the same applies hereinafter). Was dispensed at 100 μl per well.

The fused cells were in a CO ₂ incubator (5% C
(O ₂ , 37 ° C.), the medium was replaced with a HAT medium, and the cells were grown. The fused cells composed of spleen cells and myeloma cells were screened. Then, it was conditioned in HT medium, and further 10% FCS (fetal calf serum) -RPMI1
Conditioned in 640 medium.

The antibody in the culture supernatant of the fused cells was phosphorylated M
V55 peptide was detected by ELISA using a microtiter plate sensitized. For positive wells, cloning by limiting dilution was repeated twice to select one clone having reactivity to the phosphorylated MV55 peptide, and the fused cell clone 4A4
(Accession number FERM P-15759). The cells of this clone were suspended in 90% bovine serum containing 10% DMSO and stored in liquid nitrogen.

Example 4 Collection of Monoclonal Antibody The monoclonal antibody produced by the clone 4A4 was
Clones were grown in the abdominal cavity of BALB / c mice, and each was purified from the ascites fluid using a protein-A Sepharose 4B column.

[Example 5] Confirmation of antibody specificity The monoclonal antibody obtained in Example 4 was subjected to ELISA
MV5 by Western blotting and Western blotting
Reactivity with 5 peptides was confirmed. 1) Confirmation of specificity by ELISA method The monoclonal antibody obtained in Example 4 was added to each of the microtiter plate to which the MV55 peptide was bound and the microtiter plate to which the phosphorylated MV55 peptide was bound, followed by reaction. After washing, a peroxidase-labeled anti-mouse IgG (manufactured by Medical Biology Laboratories) was reacted, washed again, and reacted with a solution of hydrogen peroxide and orthophenylenediamine as a substrate, and the absorbance of the substrate was measured. As a result, the monoclonal antibody obtained in Example 4 reacted with the phosphorylated MV55 peptide, but did not react with the MV55 peptide.

2) Confirmation of Specificity by Western Blot Mouse vimentin phosphorylated with cdc2 kinase, cAMP-dependent protein kinase, protein kinase C and Ca ⁺⁺ -calmodulin-dependent protein kinase II, and non-phosphorylated mouse vimentin SDS polyacrylamide gel electrophoresis was performed on mouse vimentin, and Western blot was performed using the monoclonal antibody obtained in Example 4. As a result, only mouse vimentin phosphorylated by cdc2 kinase showed reactivity, and non-phosphorylated vimentin and vimentin phosphorylated by other protein kinases did not show reactivity.

3) Confirmation of Specificity by Inhibition Assay 75 ng of mouse vimentin phosphorylated by cdc2 kinase was applied to each lane of SDS polyacrylamide gel electrophoresis, and PBS, 50 μg / ml phosphorylated MV55 peptide and 50 μg / ml ml of non-phosphorylated MV
Western blot was performed using the monoclonal antibody of Example 4 (1 μg / ml) absorbed with 55 peptides. As a result, a vimentin band was confirmed when the monoclonal antibody absorbed with PBS and the non-phosphorylated MV55 peptide was used, but a vimentin band was not confirmed when the antibody was absorbed with the phosphorylated MV55 peptide. From the above, it was confirmed that the antibody produced from clone 4A4 obtained in Example 4 specifically reacted with the phosphorylated MV55 peptide, and was confirmed to be an anti-phosphorylated MV55 peptide antibody. The specificity of the antibody produced from clone 4A4 was determined according to The Journal of Biological Chemistry.
al Chemistry) Vol. 269, No. 49, pp. 31097-31106 (1994).

Example 6 Construction of Measurement System Using the monoclonal antibody obtained in Example 4, a system for measuring the activity of protein kinase in a sample was constructed as follows.

[6-1] Preparation of Immobilized Antibody The monoclonal antibody obtained in Example 4 (anti-phosphorylated MV5
5 peptide antibody) in 0.1 M carbonate buffer pH 9.0.
It was adjusted to a concentration of μl / ml, and 100 μl was added to each well of a 96-well Nunc Microplate “Maxisorp”, and allowed to react at 4 ° C. for 20 hours. The antibody solution was discarded, and 200 μl of PBS containing 1% BSA and 5% sucrose was added to each well, and the mixture was allowed to stand at room temperature (20 to 25 ° C.) for 2 hours to perform blocking. Discard the blocking solution,
The plate was air-dried to obtain an immobilized antibody (antibody-sensitized plate) in which an anti-phosphorylated MV55 peptide antibody was bound to an insoluble support. This immobilized antibody was sealed and stored with a desiccant.

[6-2] Preparation of Biotin-Labeled MV55 Peptide The MV55 peptide was prepared by a solid phase method using the Fmoc method using a fully automatic synthesizer (for details of the synthesis method, see Shujunsha, Cell Engineering Supplement “ Anti-Peptide Antibody Experimental Protocol "
reference). The MV55 peptide was dialyzed against a 0.1 M carbonate buffer to a concentration of 5 mg / ml, and the mixture was slowly stirred with a stirrer.
0.1 of S-LC-BIOTIN (manufactured by PIERCE)
M carbonate buffer (pH 8.5) was added so that the molar ratio of peptide to biotin was 1:20, and PBS was added.
Dialyzed to prepare a biotin-labeled MV55 peptide.

[6-3] Preparation of Specimen M-phase Hela cells were treated with 0.05 M NaCl, 1 mM
PMSF, 50 μg / ml Leupeptin, 2 m
g containing EGTA, 1 mM MgCl ₂ , 25 mM β-glycerophosphate, 10 mM 2-mercaptoethanol, 0.5 mM dithiothreitol and 0.01% polyoxyethylene (23) lauryl ether
10 mM with 0 mM Tris-HCl buffer (pH 7.5)
^It was adjusted to ⁶ cells / ml and crushed with ultrasonic waves to obtain a cell extract.

[6-4] Measurement 250 mM HEPES buffer (pH 7.5), 100 mM
mM MgCl ₂ , 10 mg / ml biotin-labeled MV
6 μl each of 55 peptide and cell extract and 30 μl of purified water
1 mM ATP was added to the mixture, and the mixture was reacted at 30 ° C. for 30 minutes.
The reaction was stopped by adding 60 μl of EDTA. 100 μl of this reaction solution was added to the well of the immobilized antibody,
After reaction for 60 minutes in 0.01M PBS (pH
After washing 3 times with 7.2), 100 μl of peroxidase-labeled streptavidin (Tago, diluted 1/10000)
Was added and reacted at 25 ° C. for 60 minutes. After washing again, 100 μl of orthophenylenediamine coloring substrate (manufactured by Medical Biology Laboratories Co., Ltd.) was added to develop color, and 0.15 MH was added.
_The reaction was stopped with 100 μl of ₃ PO ₄ solution, and the wavelength was 492 nm.
Was measured.

[6-5] Comparison with Conventional Method In order to confirm the sensitivity of the new measurement method, the results were compared with the measurement results obtained by the conventional RIA method and the conventional ELISA method.

[6-5-1] Conventional RIA method 250 mM HEPES buffer (pH 7.5), 100
Sample solution containing mM MgCl ₂ , 10 mg / ml MV55 peptide and cdc2 kinase (see [6-2] above)
5 μl of each sample and 25 μl of purified water were mixed,
1 mM γ- ³² P-ATP (500,000 cpm /
5 μl) and react at 30 ° C. for 30 minutes.
μl of a 300 mM H ₃ PO ₄ solution or 20 to 10
The reaction was stopped by adding 0 mM EDTA. 50 of the reaction solution
μl spotted on p-81 cellulose filter paper, 75 mM
After washing three times with H ₃ PO ₄ , the radioactivity of the spot was measured.

[6-5-2] Conventional ELISA Method The MV55 peptide was dissolved in a physiological phosphate buffer (PBS) to prepare a 1 μg / ml solution. This liquid 100μ
was added to each well of a 96-well microtiter plate, and allowed to stand still at 4 ° C. for 12 to 18 hours. Thereafter, the peptide solution was removed, and 5% bovine serum albumin and 5%
PB containing sucrose and 0.1% sodium azide
After adding 350 μl of S, the mixture was allowed to stand at 4 ° C. for 12 to 18 hours to block unreacted sites on the microtiter plate, discard the blocking solution, and wash three times with PBS to obtain an immobilized MV55 peptide. The sample prepared in the above [6-2] is added to the immobilized MV55 peptide, reacted at room temperature for 5 to 20 minutes, and the phosphorylation reaction is stopped by adding 100 μl of a 20% phosphoric acid solution.
Washing was performed once (this immobilized the immobilized MV55 peptide).

A biotin-labeled monoclonal antibody was prepared as follows. That is, 0.1 M carbonate buffer (pH
8.5) dissolved in monoclonal antibody (clone 4A)
Antibody produced from No. 4, 5 mg / ml) and NHS-LC
-BIOTIN (25 mg / ml, manufactured by PIERCE)
Has a molar ratio of IgG to NHS-LC-BIOTIN of 1:
The mixture was added to 60 and stirred at room temperature for 4 hours using a stirrer. After stirring, the solution was dialyzed against a physiological phosphate buffer (PBS) to obtain a biotin-labeled monoclonal antibody.

Then, 100 μl of the biotin-labeled monoclonal antibody was added to the immobilized MV55 peptide phosphorylated as described above, reacted at room temperature for 60 minutes and washed, and then 100 μl of a streptavidin-POD complex solution was added.
Was added. After further reacting at room temperature for 60 minutes, washing, adding 100 μl of a mixed solution of orthophenylenediamine and hydrogen peroxide, reacting at room temperature for 3 to 5 minutes, and after developing color, adding 100 μl of a 20% phosphoric acid solution and reacting Stopped. The absorbance at a wavelength of 492 nm was measured for the colored solution.

[6-5-3] Comparison FIG. 1 shows the measurement results of the present measurement method, the conventional RIA method, and the conventional ELISA method. As is clear from FIG. 1, the sensitivity of this measurement method is about six times that of the conventional ELISA method, and a sensitivity close to that of the conventional RIA method can be obtained.

[Others] Fused cell clone 1E9 (Accession number: FERM P-15593) and a phosphorylated MEK-1 peptide capable of being phosphorylated by raf-1 kinase were used as an immunogen in the same procedure as in Examples 1-4. Clone 2F11 (Accession number: FERMP-15594)
And anti-phosphorylated MEK- was prepared in the same manner as in Example 5.
The specificity of one peptide antibody was confirmed.

Further, Examples 1 to 4 were performed using a phosphorylated PS peptide which can be phosphorylated by PKC or PKA as an immunogen.
Fusion cell clone 2B9 (Accession number: FERM P-15133) and clone 2G3
(Accession number: FERM P-15134) was obtained, and the specificity of the anti-phosphorylated MEK-1 peptide antibody was confirmed by the same procedure as in Example 5.

[0047]

According to the measurement method and the measurement kit of the present invention, the activity of various protein kinases, particularly the kinase activity of cdc2 kinase, whose Km value is not so large, can be measured easily and with high sensitivity. If it becomes possible to measure the kinase activity of various protein kinases in this way, it will provide a means for elucidating the mechanism of information transmission in cells, and at the same time, diseases that are regarded as abnormalities in the information transmission mechanism ( (E.g., cancer and some diabetes).

[0048]

[Sequence list] SEQ ID NO: 1 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Gln Leu Ile Asp Ser Met Ala Asn Ser Phe Val Gly Thr Arg Cys 1 5 10 15

SEQ ID NO: 3 Sequence length: 14 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Arg Phe Ala Arg Lys Gly Ser Leu Arg Gln Lys Asn Val Cys 1 5 10

[Brief description of the drawings]

FIG. 1 is a graph showing a comparison of sensitivity of various measurement methods.

Claims (8)

[Claims] 1. A method for measuring protein kinase phosphorylase activity, comprising the following steps (a) to (c): (A) reacting, in a liquid phase, a labeled substrate peptide obtained by labeling a substrate peptide that can be phosphorylated by protein kinase with a labeling substance, and the protein kinase;
A step of preparing a phosphorylated label substrate peptide. (B) an immobilized antibody obtained by binding an antibody that specifically binds to a phosphorylated substrate peptide (a predetermined amino acid in the amino acid sequence of the substrate peptide is phosphorylated) to an insoluble support; A) reacting the phosphorylated label substrate peptide obtained in the step 2) to bind them. (C) a step of measuring a labeling amount of a conjugate of the immobilized antibody obtained in the above (b) and the phosphorylated label substrate peptide. 2. The protein kinase is cdc2 kinase, the substrate peptide is an MV55 peptide (peptide represented by SEQ ID NO: 1 in the sequence listing), and the phosphorylated substrate peptide is a phosphorylated MV55 peptide (SEQ ID NO: 1 in the sequence listing). The serine at amino acid number 6 of the peptide represented by SEQ ID NO: 1 is phosphorylated), and the antibody is an anti-phosphorylated MV55 peptide antibody. 3. The phosphorylated anti-MV55 peptide antibody is a fused cell clone 4A4 (accession number: FERMP P).
3. The method according to claim 2, wherein the monoclonal antibody is a monoclonal antibody produced by the method described in (1). 4. The method according to claim 1, wherein the labeling substance is one selected from the group consisting of biotin, peroxidase, β-D-galactosidase, alkaline phosphatase and microperoxidase. The measurement method described in Crab. 5. A labeled substrate peptide obtained by labeling a substrate peptide that can be phosphorylated by a protein kinase with a labeling substance, and a phosphorylated substrate peptide (a predetermined amino acid in the amino acid sequence of the substrate peptide is phosphorylated). A kit for measuring a protein kinase phosphorylase activity, comprising: a solid-phased antibody obtained by binding an antibody capable of binding to an insoluble support. 6. The protein kinase is cdc2 kinase, the substrate peptide is an MV55 peptide (peptide represented by SEQ ID NO: 1 in the sequence listing), and the phosphorylated substrate peptide is a phosphorylated MV55 peptide (SEQ ID NO: 6. The measurement kit according to claim 5, wherein the serine at amino acid number 6 of the peptide represented by SEQ ID NO: 1 is phosphorylated), and the antibody is an anti-phosphorylated MV55 peptide antibody. 7. The anti-phosphorylated MV55 peptide antibody is a fused cell clone 4A4 (accession number: FERMP P).
The measurement kit according to claim 6, wherein the kit is a monoclonal antibody produced by -15759). 8. The method according to claim 5, wherein the labeling substance is one selected from the group consisting of biotin, peroxidase, β-D-galactosidase, alkaline phosphatase and microperoxidase. The measurement kit according to any one of the above.