JP3488767B2 - Monoclonal antibody, fusion cell producing the same, and method and kit for immunoassay of protein kinase activity using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that specifically binds to a phosphorylated PS peptide, a fused cell producing the same, and an immunological assay method and assay for protein kinase activity using the monoclonal antibody. Regarding the kit.

[0002]

2. Description of the Related Art Higher organisms are a group of cells that are diversely differentiated, and each cell regulates its own behavior by recognizing surrounding conditions. It is thought that this is because substances secreted by other cells or substances expressed on the surface of other cells bind to their specific receptors, and the information is transmitted into the cells.

When cells start to grow by stimulation of growth factors, information from the outside is transmitted to the inside of the cell through the cell membrane, and further, it is transmitted from the cytoplasm to the nucleus to synthesize a new protein. Begins. The process of transmitting extracellular information to the cell nucleus is called intracellular signal transduction, and it is known that protein phosphorylation and dephosphorylation play a major role in intracellular signal transduction.

[0004] Protein kinase C (PKC) and cyclic AMP-dependent protein (PKA) are also protein kinases responsible for such intracellular signal transduction, and are indispensable for studies of intracellular signal transduction. Has become. That is, since intracellular signal transduction is carried out by a cascade of protein phosphorylation, measuring the intracellular protein phosphorylation is indispensable for elucidating the intracellular signal transduction mechanism. On the other hand, since cancer and some diseases such as diabetes are regarded as abnormalities of intracellular signal transduction mechanism, specifically, the enzyme activity of PKA and PKC is measured as a means for clarifying the cause of these diseases. .

Conventionally, the enzyme activity of PKA and PKC has been measured by
Using histone H2B or histone IIIS, [γ-
^A method of using ³² P] ATP as a tracer was known (Biochemistry, Vol. 23, P503.
6-5041, 1984, Proc. Natl. Acad. Sci. USA, Vol.80,
P36-40, 1983). As a PKC activity measuring reagent, "PKC assay system" was sold by Amersham. However, since this method uses an RIA facility, it could not be measured at a general facility.

On the other hand, as a method that does not use radioactive materials,
The present inventors have already reported the measurement by the ELISA method using the anti-phosphorylated peptide antibody YC-10 (BBRC,
Vol.175, P1144-1151, 1991), this measurement kit was commercialized by the Medical and Biological Research Institute, Inc. and is already on sale.

[0007]

However, although the above-mentioned reagent can treat a large amount of a sample at a time without using a radioactive substance, it is not sensitive enough to measure the activity of intracellular phosphatase using actual cells as a sample. It was enough.
Therefore, the above-mentioned reagent was able to measure the phosphorylase activity of the purified fraction, but it was substantially impossible to measure it using living cells themselves.

The present invention has been made in view of the above problems, and provides a new monoclonal antibody against a phosphorylated peptide, cells producing the monoclonal antibody, and PKA and P using the monoclonal antibody.
The present invention provides an immunological measurement kit and a measurement method for sensitively measuring the enzyme activity of KC.

[0009]

In order to solve the above problems, the monoclonal antibody according to the first aspect of the present invention is a phosphorylated PS peptide (PS peptide represented by SEQ ID NO: 1 as described in claim 1. In which the serine of amino acid No. 7 is phosphorylated).

The fused cell which is the second aspect of the present invention is the second aspect.
The monoclonal antibody of claim 1 is produced as described in 1. Such fused cells are preferably those obtained by fusing antibody-producing cells obtained from a mammal immunized with a phosphorylated PS peptide and mammalian myeloma cells as described in claim 3. . Further, as described in claim 4, the fused cell clone 2B9 (accession number FERM P-15133) or the fused cell clone 2G3 (accession number FERM P-151).
34) is preferred.

A third aspect of the present invention, which is an immunological method for measuring protein kinase activity, is as described in claim 5.
It is characterized by including the following steps a) to c) and d) to f). That is, a) after reacting PKA or PKC as a standard substance with a solid-phased PS peptide obtained by binding the PS peptide represented by SEQ ID NO: 1 to an insoluble support, and b) the monoclonal antibody according to claim 1. Is reacted with a labeled monoclonal antibody labeled with a labeling substance, and c) a step of preparing a calibration curve by measuring the labeling amount of the reaction product, and d) reacting the sample with the immobilized PS peptide. After that, e) a labeled monoclonal antibody obtained by labeling the monoclonal antibody according to claim 1 with a labeling substance is reacted, and f) the labeled amount of this reaction product is measured, and PKA or PKC contained in the sample is obtained from the calibration curve. Measuring the enzyme activity of.

Here, as described in claim 6, the labeled monoclonal antibody is preferably a monoclonal antibody produced by the fused cell according to any one of claims 2 to 4, which is labeled with a labeling substance. In addition, claim 7
As described above, the labeling substance is preferably selected from the group consisting of biotin, peroxidase, β-D-galactosidase, alkaline phosphatase and microperoxidase.

An immunological assay kit for protein phosphatase activity, which is the fourth aspect of the present invention, comprises the PS peptide represented by SEQ ID NO: 1 bound to an insoluble support, as described in claim 8. It is characterized by comprising a solid-phased PS peptide and a labeled monoclonal antibody obtained by labeling the monoclonal antibody according to claim 1 with a labeling substance. Here, as described in claim 9, the labeled monoclonal antibody is preferably one obtained by labeling the monoclonal antibody produced by the fused cell according to any one of claims 2 to 4 with a labeling substance. Further, as described in claim 10, the labeling substance is biotin, peroxidase, β
Preferred is one selected from the group consisting of -D-galactosidase, alkaline phosphatase and microperoxidase.

[0014]

DETAILED DESCRIPTION OF THE INVENTION

[Monoclonal antibody of the first aspect of the present invention and fused cell of the second aspect of the present invention] The enzyme activity of protein phosphatase is generally measured by detecting a protein phosphorylated by protein phosphatase. You can The present inventors initially confirmed that glial fiber acidic protein (GFAP), which is a constituent protein of glial filament which is an intermediate filament existing in glial cells, is PKA and PK.
Since it is a substrate protein with good C, peptides (G1 peptide) containing phosphorylation sites of these proteins were synthesized,
A method for measuring the activity of protein kinase was invented using YC-10 which is a monoclonal antibody against phosphorylated G1 peptide. However, a peptide in which alanine at amino acid number 7 of the pseudo substrate sequence (SEQ ID NO: 2) widely recognized in the PKC isotype was replaced with serine (PS
Peptide, SEQ ID NO: 1) was reported to be a very good substrate for PKC (SCIENCE Vol. 238, P172).
6- (1990)), the inventors have invented a new assay system by preparing a monoclonal antibody against phosphorylated PS peptide.

The production of this monoclonal antibody will be described below. A. Antigen As the antigen, a phosphorylated PS peptide (a serine of amino acid number 7 of the PS peptide represented by SEQ ID NO: 1 was phosphorylated) was synthesized and used as an immunogen. Since the peptide is a relatively small molecule and it is difficult to form an antibody by immunizing this peptide as it is, it is usually used as an immunogen by binding it to a carrier protein such as KLH (keyhole limpet hemocyanin) or albumin. B. As the animal immunized with the above-mentioned antigen, a mouse as well as a rat,
Hamsters and the like can also be used. Usually, the mouse is the most commonly used, and the BALB / c mouse and other strains (str
ain) mice can be used. At this time, the immunization regimen and the concentration of the antigen should be selected so that a sufficient amount of antigen-stimulated lymphocytes are formed. For example, one mouse is immunized with 25 μg of the antigen 3 times in the abdominal cavity at intervals of 2 weeks, and then 25 μg is intravenously administered. A few days after the final immunization, spleen cells for fusion are removed. C. Cell Fusion Spleens are aseptically removed from the mammalian individuals immunized as described above, and single cell suspensions are prepared therefrom. The spleen cells (antibody-producing cells) are fused with appropriate myeloma cells by using an appropriate fusion promoter. As the myeloma cells, those derived from a mammal of the same species as the immunized animal are preferable, but spleen cells of rats, hamsters and the like and mouse myeloma cells can also be fused. The preferred ratio of spleen cells to myeloma cells is in the range of about 20: 1 to about 2: 1. The use of 0.5-1.5 ml of fusion medium is suitable for about 10 ⁸ splenocytes. As a preferable fusion promoter, for example, polyethylene glycol having an average molecular weight of 1000 to 4000 can be advantageously used, but other fusion promoters known in this field (for example, Sendai virus (also known as HVJ)) can also be used. Further, 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 Supporting unfused myeloma cells with a mixture of unfused splenocytes, unfused myeloma cells and fused fused cells in another container (eg, microtiter plate) Diluted in selective medium for sufficient time to kill unfused cells (about 1
Time) incubate. The medium used is a drug resistant (eg 8-azaguanine resistant) and does not support unfused myeloma cells (eg HAT medium). Unfused myeloma cells die in this selective medium. Since these unfused splenocytes are non-neoplastic cells, they die for a certain period of time (after 1 week). On the other hand, the fused cells have the tumorigenic properties of myeloma parental cells and the properties of parental splenocytes, and thus can survive in the selective medium.

Thus, after the fused cells have been detected, an enzyme immunoassay (E
Screening is performed by nzyme Linked Immunosorbent Assay), and only fused cells producing a monoclonal antibody that specifically binds to the phosphorylated PS peptide are selected. Examples of such fused cells include fused cell clone 2B9 (accession number FERM P-15133) or fused cell clone 2G3 (accession number FERM P-151).
34). F. Obtaining the Monoclonal Antibody of Interest After cloning the fused cells producing the monoclonal antibody of interest by an appropriate method (eg, the limiting dilution method), the antibody can be produced by two different methods. According to the first method, the fused cells are cultured for a certain period of time in an appropriate medium, whereby the monoclonal antibody produced by the fused cells can be obtained from the culture supernatant. According to the second method, the fused cells can be injected into the abdominal cavity of an immunized animal having an isogenic gene or a semi-isogenic gene. From the blood and ascites of the host animal after a certain time,
The monoclonal antibody produced by the fused cells can be obtained. [The third immunological assay method of the present invention] A. The activity of the immobilized PS peptide protein phosphatase is determined by measuring the amount of protein phosphorylated by the enzyme.
Various proteins and peptides can be considered as phosphorylation substrates, but the PS peptide (SEQ ID NO: 1) obtained by substituting serine for alanine at amino acid number 7 of the pseudo substrate sequence (SEQ ID NO: 2) widely found in PKC has a Km for PKC. Since the value (Michaelis constant) is the smallest, high sensitivity can be obtained when this is selected as a substrate and measured in combination with a monoclonal antibody against the phosphorylated PS peptide. The PS peptide is bound to the insoluble support by contacting the solution of the PS peptide with the insoluble support to adsorb the PS peptide on the surface of the insoluble support, or by a chemical method such as covalent bonding. be able to.

Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran,
Examples thereof include polymers such as polysaccharides, paper, glass, metal, agarose, and combinations thereof. Further, the shape of the insoluble carrier can be various shapes such as a tray shape, a spherical shape, a rod shape, a fiber shape, a disk shape, a container shape, a cell, a test tube and the like. B. Labeled monoclonal antibody The monoclonal antibody against the phosphorylated PS peptide is
The phosphorylated PS peptide can be produced from a fused cell obtained by fusing an antibody-producing cell obtained from a mammal immunized with a mammalian myeloma cell line. For example, fused cell clone 2B9 (accession number FERM P
15133) or the fused cell clone 2G3 (accession number FERM P-15134) can be used, but higher sensitivity can be obtained when the monoclonal antibody produced from clone 2B9 is used. . The antibody may be a whole antibody or a fragment such as Fab or F (ab) ′ ₂ .

The labeling substance is not particularly limited as long as it can be used in a usual immunological measuring method, but an enzyme, avidin, a fluorescent substance, a luminescent substance, a radioactive substance and the like are used. preferable. As enzymes, peroxidase, β-D-galactosidase, alkaline phosphatase, microperoxidase, as fluorescent substances fluorescein isothiocyanate, phycobiliprotein, phycoerythrin, etc., as luminescent substances, isorcinol, lucigenin, etc., and as radioactive substances Can be ¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H, and the like.
When biotin is used as the labeling substance, high sensitivity can be obtained by using enzyme-labeled avidin, which is more preferable. As the labeling enzyme in this case, an enzyme similar to the enzyme used for labeling the antibody can be used, but peroxidase is particularly preferable.

When an enzyme is used as a labeling substance, a substrate and optionally a color-developing agent are used to measure its activity. When using peroxidase as the enzyme,
H ₂ O ₂ was used as a substrate, and 2,2′-azino-di- [3-ethylbenzthiazolinesulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, o was used as a color-developing agent.
-Phenylenediamine (OPD), 4-aminoantipyrine, 3,3 ', 5,5'-tetramethylbenzidine, etc. When alkaline phosphatase is used as the enzyme, o-nitrophenyl phosphate is used as the substrate, and β-D is used as the enzyme. -When using galactosidase, fluoroscein-di- (β-D-galactopyranoside) as a substrate,
4-Methylumbelliferyl-β-D-galactopyranoside and the like can be used. C. Preparation of calibration curve PKA or PK as a standard substance for immobilized PS peptide
React C. The PS peptide is phosphorylated according to the amount of the standard substance used at this time to become a phosphorylated PS peptide. Next, the phosphorylated PS peptide produced is reacted with a labeled monoclonal antibody. Then, the labeled monoclonal antibody specifically binds to the phosphorylated PS peptide. Then, the labeled amount of the labeled monoclonal antibody bound to the phosphorylated PS peptide is measured. As a result, the relationship between the amount of the standard substance (PKA or PKC) and the labeling amount, that is, the calibration curve is obtained.

The reaction medium used in this immunological assay method is, for example, one having a pH range of 6.0 to 8.0 containing a phosphate buffer solution, a Tris hydrochloric acid buffer solution, an acetate buffer solution and the like. However, when measuring PKA activity, it is preferable to contain cAMP, and when measuring PKC activity, calcium ion is preferably contained. D. Measurement of the phosphorylase activity of the sample The sample is reacted with the immobilized PS peptide. PS depending on the amount of phosphorylase contained in the sample used at this time
The peptide is phosphorylated to a phosphorylated PS peptide. Next, the phosphorylated PS peptide produced is reacted with a labeled monoclonal antibody. Then, the labeled monoclonal antibody specifically binds to the phosphorylated PS peptide.
Then, the labeled amount of the labeled monoclonal antibody bound to the phosphorylated PS peptide is measured. The phosphorylase activity in the sample is measured by comparing this measured value with a calibration curve. The reaction medium is described in the above C. As described in. [Fourth Immunological Assay Kit of the Present Invention] The immunological assay kit comprises at least (1) a PS peptide bound to an insoluble carrier and (2) a monoclonal antibody that specifically binds to a phosphorylated PS peptide. Labeled monoclonal antibody labeled with a labeling substance, and may further include a reaction medium, or may include a standard substance (PKA or PKC). When the labeling substance of the labeled monoclonal antibody is an enzyme, it usually contains a substrate for measuring the activity of the enzyme and a reaction stop solution. Since the insoluble carrier and the labeling substance have been described above, the details are omitted.

According to this immunological assay kit, the P
The phosphorylase activity can be measured by reacting the S peptide with the sample, then reacting the reaction product with the labeled monoclonal antibody, and then measuring the amount of the labeling substance. In addition to the third immunological measurement method of the present invention in which the phosphatase activity is quantitatively measured by a calibration curve, this kit can also be used for qualitatively measuring the phosphatase activity, for example.

[0022]

The preferred embodiments of the present invention will be described below.
It is needless to say that the embodiment of the present invention is not limited to the following examples, and various forms can be adopted as long as they are within the technical scope of the present invention. [Example 1] Preparation of immunogen Phosphorylated PS peptide (PS peptide of SEQ ID NO: 1 in which serine at amino acid number 7 is phosphorylated) was used as an ordinary peptide synthesizer (for example, manufactured by Perkin Elmer Japan Co., Ltd.). It was obtained by synthesizing A431) using serine which was phosphorylated in advance. In addition, PSC synthesized using a peptide synthesizer is used as PKC or PKA.
It can also be obtained by phosphorylating serine with.

This phosphorylated PS peptide was bound to KLH by the MBS method to prepare an immunogen. That is, 4 mg of K
LH was added to 0.25 ml of 10 mM phosphate buffer (pH
7.2), and to this solution was added 0.7 mg of 3-maleimidobenzic acid-N-hydroxysuccinimide ester (MBS) dissolved in 20 μl of dimethylformamide (DMF), and the mixture was stirred at room temperature for 30 minutes. Let react for minutes. After the reaction, gel filtration was performed using a column (1.5 × 10 cm) packed with Sephadex G25 to obtain KLH.
-MB complex and free KLH were separated and KLH-MB complex was collected. Then, 5 mg of the phosphorylated PS peptide was dissolved in 1 ml of 0.1 M borate buffer (pH 9.0), and this solution was mixed with the KLH-MB complex solution to adjust the pH.
Was adjusted to 7.0 to 7.5 and left to react at room temperature for 3 hours. After the reaction, the mixture was thoroughly dialyzed against PBS, adjusted to a concentration of 0.5 mg / ml with PBS, dispensed in 100 µl aliquots, frozen at -20 ° C and stored. [Example 2] Immunization of mouse Synthetic peptide-bonded KLH solution 1 cryopreserved in Example 1
A suspension was prepared by thoroughly mixing 00 μl and 100 μl complete Freund's adjuvant, and this suspension was administered to the abdominal cavity of two mice (female, BALB / c) at 25 μg each as an antigen. The same amount of antigen was administered 5 times every other week, and 3 days later, the spleen was taken out and cell fusion was carried out as shown in Example 3. Example 3 Cell Fusion and Selection and Acquisition of Fused Cells Producing Monoclonal Antibody of Interest About the extracted mouse spleen cells and syngeneic mouse myeloma cells (SP-2 / 0-Ag-14) The mixture was mixed at a ratio of 10: 1, and cell fusion was performed using 50% polyethylene glycol 4000 as a fusion promoter. 1x1 cells after fusion
The cells were suspended in HAT medium containing 10% bovine serum so as to have a cell concentration of 0 ⁶ cells / ml, and 100 μl was dispensed to each well of a 96-well microtiter plate (maximum soap manufactured by Nunc, the same applies below).

The fused cells were incubated in a CO ₂ incubator (5%
(CO ₂ , 37 ° C.), the medium is exchanged with a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium), and the cells are grown in HAT medium to produce fused cells consisting of spleen cells and myeloma cells. Screened. Then acclimate in HT medium and further 10% FCS-RPM
I1640 medium was conditioned.

The antibody in the fused cell culture supernatant was phosphorylated P
The detection was performed by the ELISA method using a microtiter plate on which the S peptide was immobilized. For positive wells, cloning by the limiting dilution method was repeated twice, and two clones having reactivity with phosphorylated PS peptide were selected.
9 and 2G3 (accession number FERM P-15133, F
ERM P-15134). The obtained clones 2B9 and 2G3 were suspended in 90% bovine serum containing 10% DMSO and stored in liquid nitrogen. The monoclonal antibody produced by each clone was obtained by growing the clone in the abdominal cavity of BALB / c mice and purifying each from the ascites using a Protein-A Sepharose 4B column. [Example 4] Confirmation of specificity of monoclonal antibody (4-1) Confirmation of reactivity to phosphorylated PS peptide by ELISA method Two types of monoclonal antibodies produced from the fused cell clones 2G3 and 2B9 obtained in Example 3 About antibodies, E
The specificity of the reaction to the phosphorylated PS peptide was confirmed by the LISA method.

As the solid phase, a microtiter plate on which phosphorylated PS peptide is immobilized, a microtiter plate on which non-phosphorylated PS peptide is immobilized, and a control peptide (amino acid of MIPP4 peptide represented by SEQ ID NO: 3) The microtiter plate on which the phosphorylated MIPP4 peptide obtained by phosphorylating the serine of No. 10 was immobilized was used (for the production method thereof, see the production method of immobilized PS peptide of Example 5). .

As the specimen, the monoclonal antibody produced by the clone 2G3, the monoclonal antibody produced by the clone 2B9, and the monoclonal antibody YC-1 which specifically binds to the phosphorylated G1 peptide as a control.
0 (manufactured by Medical Biology Research Institute, Inc.) was used.

Dilution series of each monoclonal antibody (10 μm
(diluted 20-fold from g / ml in order), and 100 μl of this solution was added to each well of the above-mentioned three types of solid-phased microtiter plates, reacted for 1 hour at room temperature, and then the solution was discarded. Washed 3 times with PBS. 100 μl of peroxidase-labeled anti-mouse IgG (manufactured by Medical and Biological Laboratories Co., Ltd.) diluted to a constant concentration was added to each well, reacted at room temperature for 1 hour, washed with PBS, and then washed with hydrogen peroxide and orthophenylenediamine. The solution was added and a color reaction was performed to confirm the reactivity with the phosphorylated PS peptide. The results are shown in Table 1 below and FIGS. In Table 1 and FIGS. 1 to 3, “IgG concentration” represents the concentration of the monoclonal antibody, “2G3” represents the monoclonal antibody produced by clone 2G3, and “2B9” represents the monoclonal antibody produced by clone 2B9. Represents an antibody.

[0029]

[Table 1]

From the above results, the fused cell clone 2G3
And the monoclonal antibodies produced from 2B9 show reactivity only to phosphorylated PS peptide and
There was no reactivity with the peptide or control peptide. In addition, the control monoclonal antibody YC-10 against the phosphorylated G1 peptide did not show reactivity with any of the peptides. From this, it was confirmed that both monoclonal antibodies specifically react with the phosphorylated PS peptide. (4-2) Confirmation of specificity by immunoblotting Further, the reactivity of the monoclonal antibody produced from each of the clones 2G3 and 2B9 was confirmed by an immunoblotting method using mouse brain extract by a conventional method. The result is shown in Figure 4.
Shown in. In addition, in FIG. 4, "2G3" represents the monoclonal antibody produced from clone 2G3, and "2B9" represents the monoclonal antibody produced from clone 2B9. When investigating PKC activity, Ca ²⁺ is usually used.
And phosphatidylserine (PS) were added
In order to activate KC, the case where these reagents were added was also examined (when EGTA was added, Ca in the sample was
²⁺ becomes a complex and PKC is not activated).

The following points were made clear from FIG. That is,
The monoclonal antibody produced by clone 2B9 does not react with the protein in mouse brain extract, the monoclonal antibody produced by clone 2G3 is 20 kD
Reactivity with a little smaller protein was observed, anti-P
Since the band was recognized by the KC monoclonal antibody, PKC was certainly present in the mouse brain extract (PS was detected when the anti-PKC monoclonal antibody was reacted).
And two bands were observed in the presence of Ca ²⁺ , the calcium-dependent proteolytic enzyme in the sample was activated to decompose the PKC molecule, and the regulatory domain of the PKC molecule reacted with the anti-PKC monoclonal antibody. (Probably because of this).

From the above results, when the mouse brain extract is used as a sample, the monoclonal antibody produced by clone 2G3 reacts with proteins other than phosphorylated PS peptide and affects the measurement accuracy of phosphorylated PS peptide. Is concerned. Therefore, it was decided to use the monoclonal antibody produced by clone 2B9 for the measurement system in ELISA. [Example 5] Construction of measurement system A system for measuring the activity of protein phosphatase in a sample using a monoclonal antibody that specifically binds to phosphorylated PS peptide was constructed as follows.

PS peptide was added to physiological phosphate buffer (PB
It was dissolved in S) to prepare a 1 μg / ml solution. 100 μl of this solution was added to each well of a 96-well microtiter plate and allowed to stand at 4 ° C. for 12 to 18 hours. Then, the peptide solution was removed, and 5% bovine serum albumin, 5% sucrose and 0. 350 μl of PBS containing 1% sodium azide was added, and the mixture was allowed to stand at 4 ° C. for 12 to 18 hours to block unreacted sites of the microtiter plate, discard the blocking solution, and wash 3 times with PBS to immobilize. The PS peptide was obtained.

On the other hand, purified PKA was treated with 3 mM MgCl 2.
₂ , 0.1 mM ATP, ₂ μM cAMP, 0.5 mM
Diluted with 25 mM Tris-hydrochloric acid buffer containing EDTA, 1 mM EGTA and 5 mM 2-mercaptoethanol to prepare PKA solutions having the concentrations shown in Table 2.

Then, 100 μl of each concentration of PKA solution was added to the immobilized PS peptide and reacted at room temperature for 5 to 20 minutes, 100 μl of 20% phosphoric acid solution was added to stop the phosphorylation reaction, and 5 times with PBS. Washed (this causes the immobilized PS peptide to be phosphorylated).

The biotin-labeled monoclonal antibody was prepared as follows. That is, 0.1M carbonate buffer (pH
8.5) dissolved in monoclonal antibody (2B9, 5m
g / ml) and NHS-LC-BIOTIN (PIERC
25 mg / ml manufactured by Company E) is used as IgG and NHS-LC-B.
IOTIN was added at a molar ratio of 1:60 and stirred at room temperature for 4 hours using a stirrer. After stirring, this solution was dialyzed against a physiological phosphate buffer (PBS) to obtain a biotin-labeled monoclonal antibody.

Then, after the immobilized PS peptide was reacted with PKA, biotin-labeled monoclonal antibody 100 μ
1 was added, the mixture was reacted at room temperature for 60 minutes and washed, and then 100 μl of streptavidin-POD complex solution 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 developing color, then 20%
The reaction was stopped by adding 100 μl of phosphoric acid solution. The absorbance at a wavelength of 492 nm was measured for the colored solution.

As a comparative product, G1 peptide (substrate)
Immobilized G1 peptide obtained by binding to a microtiter plate, and a peroxidase-labeled YC-labeled with peroxidase-labeled monoclonal antibody YC-10 that specifically binds to phosphorylated G1 peptide.
10 was used to measure the PKA solution of each concentration, and the sensitivity was compared with that of this example. The results are shown in Table 2 and FIG.

[0039]

[Table 2]

From the above results, it was shown that the reagent of this example can detect the protein kinase activity with extremely high sensitivity as compared with the conventional reagent (comparative product). [Example 6] Confirmation of reactivity In order to confirm that the measurement reagent specifically captures PKA and PKC activities, COS cells and Molt3 were confirmed.
Phosphorylation activity was measured using cell extracts. That is, an extract of COS cells and Molt3 cells was used as a sample. In addition, when examining PKA activity, cAM
When P (activity that activates PKA) is added or not, while PKC activity is examined, Ca
²⁺ and PS (these activate PKC) or EGTA (which chelate Ca ²⁺ in the sample) were added and the activity was measured as in Example 5. The results are shown in Tables 3 and 4 and FIGS. In these tables and figures, "protein concentration" represents the concentration of the extract of COS cells or Molt3 cells.

[0041]

[Table 3]

[0042]

[Table 4]

From the above results, the reagents are PKA and P
It was shown that the activity of these enzymes was specifically measured in all of the KC activity measurements. The control example (cA
Reactivity was also seen with MP (-) and EGTA (+) because PKA that was already activated in the cell extract
Or PKC, or a protein kinase other than PKA or PKC.

[0044]

INDUSTRIAL APPLICABILITY According to the present invention, there are provided an immunological assay method capable of assaying the activity of protein kinase in a sample with high sensitivity, and a kit therefor. It has been difficult to measure the activity of protein kinases in cells in the past, but by sensitively measuring the activities of these enzymes in unadjusted specimens, it was possible to elucidate the mechanism of intracellular signal transduction. At the same time that the means are provided
A means for elucidating the cause of a disease (cancer, some diabetes, etc.) that is regarded as an abnormality in the information transmission mechanism is provided. In addition, useful monoclonal antibodies used for these are provided,
Further provided are useful fused cells which produce this monoclonal antibody.

[0045]

[Sequence list]

SEQ ID NO: 1 Sequence length: 14 Sequence type: Amino acid Tobology: linear Sequence type: Peptide Array   Arg Phe Ala Arg Lys Gly Ser Leu Arg Gln Lys Asn Val Cys    1 5 10

SEQ ID NO: 2 Sequence length: 14 Sequence type: Amino acid Tobology: linear Sequence type: Peptide Array Arg Phe Ala Arg Lys Gly Ala Leu Arg Gln Lys Asn Val Cys  1 5 10

SEQ ID NO: 3 Sequence length: 15 Sequence type: Amino acid Tobology: linear Sequence type: Peptide Array Lys Arg Arg Leu Asn Arg Glu Glu Ser Ser Val Val Leu Gly Tyr  1 5 10 15

[Brief description of drawings]

FIG. 1 Ig with non-phosphorylated PS peptide
It is a graph showing the relationship of the light absorbency with respect to G (monoclonal antibody) density | concentration.

FIG. 2 IgG using phosphorylated PS peptide
It is a graph showing the relationship of the absorbance with respect to (monoclonal antibody) concentration.

FIG. 3: IgG when using a control peptide
It is a graph showing the relationship of the absorbance with respect to (monoclonal antibody) concentration.

FIG. 4 is an explanatory view (transcription of the state of electrophoresis) showing the results of immunoblotting of various monoclonal antibodies when a mouse brain extract was used.

FIG. 5: P when using the reagents of Example 5 and comparative products
It is a graph showing the relationship of the light absorbency with respect to KA.

FIG. 6 is a graph showing the relationship of absorbance with respect to protein concentration in COS cells regarding PKA activity.

FIG. 7 is a graph showing the relationship between the PKA activity and the absorbance of Molt3 cells with respect to the protein concentration.

FIG. 8 is a graph showing the relationship between the absorbance of COS cells and the protein concentration of PKC activity.

FIG. 9 is a graph showing the relationship between the protein concentration of Molt3 cells and the absorbance with respect to PKC activity.

─────────────────────────────────────────────────── ─── Continuation of front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 33/53 C07K 16/40 G01N 33/577 SwissProt / PIR / GeneS eq

Claims (10)

(57) [Claims] 1. A monoclonal antibody that specifically binds to a phosphorylated PS peptide (a PS peptide represented by SEQ ID NO: 1 in which serine at amino acid number 7 is phosphorylated). 2. A fused cell producing the monoclonal antibody according to claim 1. 3. The fused cell according to claim 2, which is obtained by fusing an antibody-producing cell obtained from a mammal immunized with a phosphorylated PS peptide and a mammalian myeloma cell. 4. The fused cell clone 2B9 (accession number FE)
RM P-15133) or fused cell clone 2G3
(Accession number FERM P-15134)
The fused cell described. 5. A method for immunologically measuring protein kinase activity, which comprises the following steps a) to c) and d) to f). a) A solid-phased PS peptide obtained by binding the PS peptide represented by SEQ ID NO: 1 to an insoluble support was reacted with cyclic AMP-dependent protein (PKA) or protein kinase C (PKC) as a standard substance. Then, b) reacting the labeled monoclonal antibody obtained by labeling the monoclonal antibody according to claim 1 with a labeling substance, and c) creating a calibration curve by measuring the labeled amount of this reaction product, and d) the above After reacting the sample with the solid-phased PS peptide, e) a labeled monoclonal antibody obtained by labeling the monoclonal antibody according to claim 1 with a labeling substance is reacted, and f) the labeled amount of the reaction product is measured, and A step of measuring the enzyme activity of PKA or PKC contained in the sample from a calibration curve. 6. The immunological method according to claim 5, wherein the labeled monoclonal antibody is obtained by labeling the monoclonal antibody produced by the fused cell according to any one of claims 2 to 4 with a labeling substance. Measuring method. 7. The immunological measurement method according to claim 5, wherein the labeling substance is selected from the group consisting of biotin, peroxidase, β-D-galactosidase, alkaline phosphatase and microperoxidase. 8. A solid-phased PS peptide obtained by binding the PS peptide represented by SEQ ID NO: 1 to an insoluble support, and a labeled monoclonal antibody obtained by labeling the monoclonal antibody according to claim 1 with a labeling substance. An immunological assay kit for protein phosphatase activity, which comprises: 9. The immunological method according to claim 9, wherein the labeled monoclonal antibody is obtained by labeling the monoclonal antibody produced by the fused cell according to any one of claims 2 to 4 with a labeling substance. Measurement kit. 10. The immunological assay kit according to claim 8, wherein the labeling substance is selected from the group consisting of biotin, peroxidase, β-D-galactosidase, alkaline phosphatase and microperoxidase.