JPH085632A - Antibody for recognizing active map kinase - Google Patents

Abstract

PURPOSE:To analyze active MAP kinase conveniently and quickly with high accuracy. CONSTITUTION:An antigenic peptide containing a unique amino acid series between the subdomains VII and VIII of an active MAP kinase is bonded directly, or indirectly through a spacer, to a high molecular weight carrier molecule to produce a composite antigen for an antibody. The unique amino acid series is N-Thr (PO3H2)-Glu-Tyr (PO3H2)-C reacts on the active MAP kinase under presence of 0.1%(v/v)SDC but not does not substantially react when the O. 1% (v/v) SDC IS not present. Furthermore, the unique amino acid series substantially exhibits no cross reactivity to inactive MAP kinase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody that recognizes active MAP kinase, a method for producing the same, and use thereof.

[0002]

2. Description of the Related Art Conventionally, activated MAP (Mitogen-Activated)
Protein) Kinase can be detected with various proteins that are intracellular substrates in the presence of radiolabeled phosphorus and the like.
By reacting P-kinase, the above-mentioned phosphorus and the like are incorporated into various proteins, and the phosphorylation amount in the proteins is adjusted to X
A method for examining the presence (amount) of active MAP kinase in a test MAP kinase by detecting it as a light-sensitive spot or band on a linear film has been known.

[0003]

However, since this method uses a radioisotope such as radiolabeled phosphorus, it requires labor, time and skill for sample preparation, reaction, etc., and a large-scale measurement. Equipment and special facilities were essential. Therefore, it has a drawback of lacking in simplicity. Moreover, it was not always sufficient in terms of detection sensitivity.

[0004]

In view of the above situation, the present inventors have conducted diligent studies to find an excellent method for detecting and analyzing active MAP kinase, and as a result, active MA
The inventors have found that it is possible to detect and analyze active MAP kinase very efficiently by using an antibody that recognizes P kinase, and completed the present invention. That is, the present invention provides a complex in which an antigenic peptide containing a unique amino acid sequence between subdomains VII and VIII of activated MAP kinase is directly or indirectly bound to a high molecular weight carrier molecule through a spacer. (1) a unique amino acid sequence is N
-Thr (PO3H2) -Glu-Tyr (PO3H2) -C, (2) 0.1
% (W / v) reacts with active MAP kinase in the presence of SDS and does not substantially react in the absence of (3) inactive M
An antibody recognizing active MAP kinase (hereinafter referred to as the antibody of the present invention), which is characterized by having substantially no cross-reactivity to AP kinase, and active M.
AP Kinase Subdomain VII and Subdomain VI
II. Immunizing a mammal against a complex in which an antigenic peptide containing a unique amino acid sequence between II is directly or indirectly bound to a high molecular weight carrier molecule through a spacer, and then blood is collected from the mammal. And separate it from the blood
The present invention provides a method for producing an antibody characterized by purification (hereinafter referred to as the production method of the present invention) and its use.

[0005] MAP kinase is a kind of serine / threonine kinase widely held in eukaryotes, and an enzyme that plays an important role in a pathway for transmitting a growth stimulus from outside the cell (intracellular signal transduction pathway). one of. For example, when a cell receives an external growth stimulus such as a growth factor or a hormone, the receptor recognizes the stimulus and generates a signal for activating the MAP kinase activating enzyme. The MAP kinase activating enzyme activated by this signal acts on the MAP kinase to activate the MAP kinase. Activated MAP kinase, that is, “active MAP kinase” leads cells to a proliferative state by phosphorylating various proteins that are intracellular substrates. In this way, cells respond to an external growth stimulus and initiate cell growth. Proliferation stimulation → Receptor → MAP kinase activating enzyme → [(inactive type) MAP kinase → active MA
P kinase ] → [(inactive type) intracellular substrate → active type intracellular substrate] → cell proliferation Therefore, the presence or absence of active MAP kinase is detected.
If it can be analyzed, it will be possible to determine the state of cell proliferation, and it will also be useful for early detection of cancer cells that grow indefinitely. In the present invention, an antibody that recognizes active MAP kinase is used as an excellent method for detecting and analyzing active MAP kinase. The antibody of the present invention comprises a complex in which an antigenic peptide containing a unique amino acid sequence between subdomains VII and VIII of activated MAP kinase is directly or indirectly bound to a high molecular weight carrier molecule through a spacer. An antibody obtained as an antigen,
(1) The unique amino acid sequence is N- Thr (PO3H2) -Glu-Tyr (PO3
H2) -C, (2) Reacts with active MAP kinase in the presence of 0.1% (w / v) SDS, and does not substantially react in the absence of (3) Inactive MAP kinase On the other hand, it is a novel antibody characterized by exhibiting substantially no cross-reactivity.

Subdomain VI of active MAP kinase
The amino acid sequence between I and subdomain VIII is, for example, N-Asp-His-Thr-Gly-Phe-Leu-Thr (PO3H
2) -Glu-Tyr (PO3H2) -Val-Ala-Thr-Arg-Trp-Tyr-Arg-Ala-
Pro-Glu -C and the like, and among them, a unique amino acid sequence is N-Thr (PO3H2) -Glu-Tyr (PO3H2) -C.
There is. As a peptide containing this unique amino acid sequence, for example, a peptide represented by the following amino acid sequence is considered. (1) N- Asp-His-Thr-Gly-Phe-Leu-Thr (PO3H2) -Glu-Tyr
(PO3H2) -Val-Ala -C (2) N- His-Thr-Gly-Phe-Leu-Thr (PO3H2) -Glu-Tyr (PO3H
2) -Val-C (3) N- Thr-Gly-Phe-Leu-Thr (PO3H2) -Glu-Tyr (PO3H2) -V
al-Ala -C (4) N- Gly-Phe-Leu-Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-C (5) N- Gly-Phe-Leu-Thr (PO3H2) -Glu-Tyr (PO3H2) -C (6) N- Thr (PO3H2) -Glu-Tyr (PO3H2) -C (7) N- Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala -C (8) N- Leu-Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala-Thr-
C (9) N- Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala-Thr-Arg-
C (10) N- Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala-Thr-Arg-
Trp -C (11) N- Phe-Leu-Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala-
Thr-Arg-Trp -C (12) N- Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala-Thr-Arg-
Trp-Tyr-Arg -C (13) N- Leu-Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala-Thr-
Arg-Trp-Tyr-Arg-Ala -C (14) N- Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala-Thr-Arg-
Trp-Tyr-Arg-Ala-Pro -C (15) N-Phe-Leu-Thr (PO3H2) -Glu-Tyr (PO3H2) -Val-Ala-
Thr-Arg-Trp-Tyr-Arg-Ala-Pro-Glu-C Such peptides are antigenic (epitopic)
May be any as long as it has The above peptides can be produced by polymerizing amino acids in the order of their amino acid sequences (solid phase method). For example,
It can be easily produced by using an ordinary automatic amino acid synthesizer.

The antibody of the present invention can be produced, for example, by the following method. [Complete antigen (immunogenic) process of an antigenic peptide containing a unique amino acid sequence] An antigenic peptide containing a unique amino acid sequence is directly or indirectly bound to a high molecular weight carrier molecule through a spacer to form a complex. obtain. The antigenic peptide containing the unique amino acid sequence used is preferably highly pure. Therefore, if necessary, it can be purified in advance by a usual method such as high performance liquid chromatography. The high molecular weight carrier molecule used is free to undergo a ligation reaction with an antigenic peptide containing a unique amino acid sequence and a compound in which a spacer is bound to these (hereinafter, both are collectively referred to as incomplete antigen). It may be any macromolecular compound that has a reactive group that can be utilized and that is capable of conferring immunogenicity on it by being linked to said incomplete antigen or enhancing its immunogenicity already present. Macromolecular compounds containing freely available reactive amino groups are particularly preferred. For example, a protein rich in lysine having a molecular weight of about 10,000 to about 150,000 can be mentioned. Specifically, bovine serum albumin (BSA: molecular weight 66200), human serum albumin (HSA: molecular weight 5
8000), rabbit serum albumin (RSA: molecular weight 6800
0), goat serum albumin (GSA: molecular weight 68000) or keyhole limpet hemocyanin (KLH: molecular weight> 1000000). As long as other macromolecular compounds meet the above requirements, it is possible to use them as carrier molecules, such compounds including:
For example, B2 microglobulin, hemocyanin, immunoglobulins, toxins (cholera toxin, tetanus toxin, diphtheria toxin and others), polysaccharides, lipopolysaccharides, natural or synthetic polyadenylic and polyuridylic acids, polyalanine and polylysine polypeptides, or cell membrane components such as Examples include red blood cell membranes treated with formalin or glutaraldehyde. The method of binding the incomplete antigen to the macromolecular carrier molecule described above allows the unique amino acid sequence sites in the incomplete antigen to remain freely available and thus elicit a specific immune response, i.e. Any method may be used as long as it can induce the production of specific antibody. Specifically, for example, (1) select an incomplete antigen such that the site of the unique amino acid sequence in the incomplete antigen is as outer as possible, and (2) select the unique amino acid sequence in the selected incomplete antigen. It is preferred that the site be as outer as possible from the polymeric carrier molecule. When the reactive group of the incomplete antigen is a reactive amino group, a dialdehyde, such as glutaraldehyde, is used to attach the reactive group of the incomplete antigen to one of the reactive amino groups of the high molecular weight carrier molecule. If the reactive group of the incomplete antigen is a reactive SH group, the reactive group of the incomplete antigen is attached to one of the reactive SH groups of the high molecular weight carrier molecule by an oxidation reaction. When the reactive group of the incomplete antigen is a reactive carboxyl group, for example, carbodiimide, preferably 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is used to convert the reactive group of the incomplete antigen into a high molecular weight carrier. It is attached to one of the reactive amino groups of the molecule. As a concrete example, for example, Chem. Pharm. Bull.
31 , (11), 4001-4007 (1983) and the active ester method by H. Hosoda et al. Or J. Biol. Chem., 234, 1090-
It can be produced by binding an incomplete antigen having a reactive carboxyl group to a reactive amino group of a high molecular weight carrier molecule by the mixed acid anhydride method by BF Franger et al. Described in 1094 (1959).

The spacer used when indirectly linked via a spacer is capable of forming at least one covalent bond of a freely available reactive group of a high molecular weight carrier molecule.
Compounds containing one or more reactive groups. For example, mention may be made of compounds containing between 2 and 16 crosslinkable carbon atoms and having as reactive group one or more reactive groups, such as amino, carboxyl, maleimide or SH groups. it can. Specifically, the general formula H ₂ N (CH ₂ ) n COOH (n is an integer from 2 to 16) is preferred. The spacer can be linked to the antigenic peptide containing the unique amino acid sequence by the same method as the method of binding the reactive group of the incomplete antigen to one of the reactive groups of the high molecular weight carrier molecule.

[Mammalian immunization step and step of obtaining antibody of the present invention] For the complex thus obtained, for example, mammals of mouse, hamster, guinea pig, chicken, rat, rabbit, dog, etc. To immunize animals, for example, J. ASSOC. OFF. ANAL. CHEM. 70 ( 6) 102
5-1027 (1987) and the like, and a conventional immunization method such as WH Newsome is used, for example, by one or more administrations of the complex. For example, administration of 2 or 3 times at intervals of 7 to 30 days is preferable. The dose is, for example, about 0.05 to 2 mg of the complex.
Use the degree as a guide. The routes of administration include subcutaneous administration, intradermal administration,
Although intraperitoneal administration, intravenous administration, intramuscular administration and the like can be selected, injection which can be performed intravenously, intraperitoneally or subcutaneously is a preferable administration form. Furthermore, the combination of subcutaneous injection and intraperitoneal injection is particularly preferable. In this case, the complex is a suitable buffer such as complete Freund's adjuvant (mixed with Aracel A, Bayol F, killed tuberculosis bacterium), RAS [MPL (Monophosphoryl Lipid A) + TDM (Synt
hetic Trehalose Dicorynomycolate) + CWS (Cell Wall Sk
eleton) Adjuvant system], which is used by dissolving it in a sodium phosphate buffer containing one of commonly used adjuvants such as aluminum hydroxide, physiological saline, etc., depending on the administration route, conditions, etc. In some cases, no such adjuvant is used. Here, the adjuvant means a substance that nonspecifically enhances an immune response to the antigen when administered together with the antigen. And
After leaving the above mammals without treatment for 0.5 to 4 months, a small amount of the serum of the mammals is sampled from an ear vein or the like to measure the antibody titer. When the antibody titer rises, administration of the complex is carried out an appropriate number of times depending on the situation. For example 100 μg to 1000 μg, especially 50 μg to 5
The dose of 00 μg of the complex is administered 1 to 5 times. Blood is collected from the immunized mammal 2 days or 2 months after the last administration by a conventional method,
By separating and purifying the blood by a conventional method such as centrifugation, precipitation by using ammonium sulfate or polyethylene glycol, chromatography such as gel filtration chromatography, ion exchange chromatography, affinity chromatography and the like, a polyclonal antibody is obtained. The antibody of the present invention can be obtained as serum. The antiserum may be inactivated in the complement system by treatment at 56 ° C. for 30 minutes, for example. In addition, immunocompetent B from the above immunized mammal
The cells are isolated, the immunocompetent B cells are fused with tumor cells capable of serial cell division, the resulting fusion is isolated, and after selection, the hybridoma cells producing the desired antibody are cloned. By culturing the hybridoma cells in vitro or in vivo to produce a monoclonal antibody, the antibody of the present invention having a high degree of specificity and affinity can also be produced.

In the antibody of the present invention, an antigenic peptide containing a unique amino acid sequence between subdomains VII and VIII of activated MAP kinase is directly or indirectly bound to a high molecular weight carrier molecule through a spacer. An antibody obtained by using the complex as an antigen, wherein (1) the unique amino acid sequence is N-Thr (PO3H2) -Glu-Tyr (PO3H2) -C, (2) 0.1% (w / v) Active MA in the presence of SDS
The novel antibody is characterized by reacting with P kinase and not reacting substantially in the absence thereof (3) showing substantially no cross-reactivity with inactive MAP kinase. The antibody of the present invention recognizes active MAP kinases derived from various organisms such as amphibians such as Xenopus and mammals such as rats.
"It reacts with active MAP kinase in the presence of 1% (w / v) SDS and does not substantially react in the absence of SDS." Means that the reaction system such as an aqueous solution, physiological saline, buffer solution or biological sample is terminated. When the concentration of 0.1% (w / v) SDS is included, the antibody of the present invention recognizes active MAP kinase, while
When SDS is not contained, the antibody of the present invention is active MAP.
This means that the kinase is not substantially recognized. In addition, "substantially no cross-reactivity with inactive MAP kinase" means that the cross-reactivity with a MAP kinase different from the target active MAP kinase, that is, inactive MAP kinase, is exhibited. It is meant that selective recognition is substantially possible, such as less than about 1/4, preferably less than about 1/20, and more preferably less than 1/100. Here, the degree of cross-reactivity can be determined by, for example, detecting an active MAP kinase by an immunoblot method described later, and a substrate protein concentration at which the inactive MAP kinase is detected by the same method. If the protein concentration is C, it is determined as 1 / C.

Use of the antibody of the present invention includes active MAP
Examples include a method of immunologically detecting active MAP kinase by detecting the antibody of the present invention that has acted on the kinase or the antibody against the antibody with the label of the antibody. Specific examples thereof include a method for analyzing active MAP kinase by immunoblotting, immunoprecipitation separation, indirect competitive inhibition (ELISA), cell or tissue immunological staining, and the like. . The analysis method is a simple and highly accurate analysis method that can be processed quickly. For example, the following method can be used.

I. Immunoblotting method This is a method of recognizing active MAP kinase present in a sample bound to a solid support by the antibody of the present invention (first antibody) and detecting the antibody. Nitrocellulose in the form of a membrane, sheet, filter or the like is generally used as a solid support for binding the sample, but the sample is well adsorbed and the antigenicity of the active MAP kinase present in the sample is high. There is no particular limitation as long as it does not disappear.
When using nitrocellulose, binding of the sample to nitrocellulose is accomplished by spotting the resulting solution of the sample in a suitable buffer, such as phosphate buffered saline, onto the nitrocellulose. It The amount of spots for quantification is preferably such that the amount of the antibody of the present invention (first antibody) with respect to the active MAP kinase as an antigen becomes excessive, and about 1 μl / 3 mm square is preferable. In order to recognize the active MAP kinase present in the sample by the antibody of the present invention (first antibody), the sample should be treated with 0.1% (w / v) SDS or the like in advance, or it should be bound to nitrocellulose. 0.1% (w / v) SDS in the buffer used at the time
It is necessary to include and process. The nitrocellulose spotted in this way is applied for about 1 minute to about 2
Incubate for 4 hours under appropriate humidity conditions. After the incubation, in order to prevent non-specific adsorption to a site other than the spotted site, the solid support material to which the sample is bound is treated with a high molecular weight carrier molecule which is not recognized by the antibody of the present invention (first antibody), Among the high molecular weight carrier molecules that can be used in the step of completely antigenizing (immunogenic) an antigenic peptide containing a unique amino acid sequence, a high molecular weight carrier molecule that is not used in the production of the antibody (first antibody) of the present invention (goat , A solution containing serum albumin or skim milk of another animal such as bovine, for about 20 minutes to about 24 hours,
The surface of the solid support is covered with the high molecular weight carrier molecule by incubation at room temperature. After incubation,
The obtained solid support material is thoroughly washed to remove the above-mentioned high molecular weight carrier molecules in the free state. The solid support material thus prepared is mixed with a preparation liquid containing the antibody of the present invention (first antibody), and then incubated with shaking for about 10 minutes to about 3 hours. The preparation containing the antibody of the present invention (first antibody) means that the antibody of the present invention (first antibody) is in a free state,
It refers to a preparation solution that may be present in a solution such as distilled water, a buffer solution, or a physiological saline solution. In this way, the active form M present in the sample
AP kinase is recognized by the antibody of the present invention (first antibody). Next, a method for detecting the antibody will be described. The antibody of the present invention (first antibody) is, for example, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-.
When labeled with an enzyme such as phosphate dehydrogenase, after incubation, the antibody of the present invention in a free state (first antibody) is removed by washing, and then the substrate of the above-mentioned labeled enzyme is allowed to act, The antibody of the present invention (first antibody) can be detected by measuring the reaction by color development or the like. For example, in the case of labeling with peroxidase, the combination of hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a color-developing reagent produces a brown or yellow color. When labeled with glucose oxidase, for example, 2,2′-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) or the like is used as a substrate.
When a second antibody labeled with an enzyme or the like that recognizes and binds to the antibody of the present invention (first antibody) is used, the present antibody (first antibody) in a free state after incubation
Are removed by washing and then incubated with the second antibody. The second antibody labeled with this enzyme or the like includes
For example peroxidase, alkaline phosphatase,
β-D-galactosidase, glucose oxidase,
An antibody to the first antibody (the antibody of the present invention) bound with an enzyme such as glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase can be mentioned. As a specific example, when a rabbit antiserum is used as the first antibody (antibody of the present invention), as the second antibody, peroxidase-conjugated anti-rabbit immunoglobulin (IgG) goat immunoglobulin (Ig) is used.
G) can be preferably mentioned. The anti-rabbit IgG goat IgG is commercially available and can be easily obtained. For detection of the second antibody, the same method as in the case of the labeled antibody of the present invention (first antibody) can be mentioned.

II. Separation method by immunoprecipitation The active MAP kinase present in the sample is recognized by the antibody of the present invention (first antibody), and an immune complex consisting of the antibody and the active MAP kinase is purified to obtain only the active MAP kinase from the sample. And separating the separated active MAP kinase by gel electrophoresis, enzyme activity measurement,
It is used for the detection method by a method such as immunoblotting. First, the sample and the antibody of the present invention (first antibody) against activated MAP kinase are added, for example, for about 1 hour to about 24 hours.
Immune complexes are formed by mixing for 4 hours at 4 ° C. with agitation. The mixing ratio of the sample to the antibody of the present invention (first antibody) at this time may be, for example, about 1: 8, but may be appropriately increased or decreased depending on the amount of active MAP kinase present in the sample. In addition, the sample
It is necessary to treat with 0.1% (w / v) SDS etc. Next, a secondary reagent that specifically binds to the antibody and can separate the antibody and the active MAP kinase bound to the antibody from the solution is added to the formed immune complex, and the mixture is incubated. By doing so, a complex consisting of the immune complex and the secondary reagent is formed, and this is recovered. Here, examples of the secondary reagent include a bacterial cell wall protein that binds to an antibody such as protein A and protein G, or an anti-immunoglobulin antibody. If these secondary reagents bound to an insoluble support material are used, the recovery (centrifugation, washing, etc.) of the complex consisting of the immune complex and the secondary reagent is extremely easy. Insoluble support materials have a very wide range of designs and can have very different shapes depending on the particular purpose for which they are intended. Examples thereof include beads, dishes, spheres, plates, small rods, cells, small bottles, small tubes, fibers and nets. Specific examples include beads made of polysaccharides such as agarose (eg, sepharose, biogel, etc.) and transparent plastic materials such as microtiter plates made of polyvinyl chloride or polystyrene, spheres made of polystyrene and polystyrene latex, and tubes. Alternatively, a rod or the like can be used. Active MAP kinase may be released from the recovered complex by an operation such as heat treatment. Then, the active MAP kinase in the free state or in the complex state may be detected by a method such as gel electrophoresis, enzyme activity measuring method, immunoblotting method and the like.

III. Indirect competitive inhibition method (ELISA method) Basically, competition between an antigenic peptide containing a unique amino acid sequence which is an antigen bound to a solid support material and an active MAP kinase contained in a sample, for example, a solid support material It is based on the competition between an antigenic peptide containing a unique amino acid sequence, which is an antigen bound to the antibody, and an active MAP kinase, which is a free antigen of the first antibody recognized by the labeled second antibody. In this connection, the binding of the antigenic peptide containing the unique amino acid sequence of the antigen to the solid support is carried out directly or with a spacer and / or first
It can occur via a high molecular weight carrier molecule that is not recognized by the antibody (ie the antibody of the invention). Here, the high molecular weight carrier molecule that is not recognized by the first antibody (that is, the antibody of the present invention) is a high molecular weight carrier molecule that can be used in the step of complete antigen (immunogen) formation of an antigenic peptide containing the above-mentioned unique amino acid sequence. Among the carrier molecules, it is a high molecular weight carrier molecule that is not used in the production of the first antibody. This is important when the antibody of the present invention is polyclonal. Further, when an antigenic peptide containing a unique amino acid sequence which is an antigen is bound via a spacer and / or a high molecular weight carrier which is not recognized by the first antibody, the binding involves the antigen containing the unique amino acid sequence. A method similar to or similar to the step of forming a complete antigen (immunogen) of a sex peptide can be used. Solid supports used for direct or indirect binding of antigenic peptides containing unique amino acid sequences that are antigens include, for example, plastic surfaces of microtiter plates or test tubes, polystyrene, polypropylene, polyvinyl chloride, glass or plastic. Beads surface consisting of etc.,
Mention may be made of the surface of filter paper, dextran, strips of cellulose or nitrocellulose or other similar material. An antigenic peptide containing a unique amino acid sequence as an antigen is directly bound to these solid supports or indirectly through a spacer and / or a high molecular weight carrier molecule which is not recognized by the first antibody (antibody of the present invention). In the following (hereinafter referred to as coating), for example, the solid support material is activated by a usual method using glutaraldehyde, cyanogen bromide or the like. The coating liquid used is, for example, 14
An example is a phosphate buffer (pH 7.4) of about 10 mM containing 0 mM sodium chloride. As coating conditions, for example, an antigenic peptide containing a unique amino acid sequence that is an antigen, or a spacer and / or
Alternatively, the concentration of the antigenic peptide containing a unique amino acid sequence which is an antigen having a high molecular weight carrier molecule which is not recognized by the first antibody in the coating solution is about 0 as an antigenic peptide containing a unique amino acid sequence which is an antigen. The preferred range is 0.05 μg / ml to about 1 μg / ml. The coating time may be, for example, about 6 hours to 24 hours. In addition, as a specific example of the above, for example, using a 96-well microtiter plate made of polystyrene as a solid support, a complex for producing the first antibody (an antigenic peptide containing a unique amino acid sequence An antigen containing serum albumin or keyhole limpet hemocyanin of an animal such as bovine as a high molecular weight carrier molecule used for a high molecular weight carrier molecule directly or indirectly bound to a high molecular weight carrier molecule, and containing a unique amino acid sequence The use of serum albumin from another species of animal, such as goat, which is a high molecular weight carrier molecule that is not recognized by the first antibody, is used as the high molecular weight carrier molecule that is used when indirectly binding a reactive peptide to a solid support. it can. The thus obtained antigenic peptide containing a unique amino acid sequence that directly or indirectly binds to the solid support is then added to the active MAP kinase and the first antibody (which are the antigens to be detected contained in the sample). The antibody of the present invention) is mixed with the test solution, and the mixture is incubated. The active MAP kinase, which is the antigen to be detected and is contained in the sample, is in free form at this time in an aqueous solution, physiological saline,
It may be present as a component such as a buffer or biological sample. About 1
After incubation for 0 minutes to about 2 hours, the mixture is incubated with a second antibody labeled with an enzyme or the like that recognizes and binds to the first antibody (the antibody of the present invention). Examples of the secondary antibody labeled with this enzyme or the like include peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, etc. An antibody to the first antibody (the antibody of the present invention) to which the enzyme of (1) is bound can be mentioned. As a specific example, when a rabbit antiserum is used as the first antibody (antibody of the present invention), as the second antibody, anti-rabbit immunoglobulin (IgG) and goat immunoglobulin (IgG) conjugated with peroxidase are preferred. be able to. The anti-rabbit IgG goat I
gG is commercially available and is easily available. When labeled with peroxidase, a brown or yellow color is produced by combining hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a color-forming reagent. When labeled with glucose oxidase, for example, 2,2'-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid (A
BTS) or the like is used.

The present invention also includes a kit for immunological detection and analysis of active MAP kinase. The above-mentioned test kit may contain the following components, for example: (1) Subdomain VII and subdomain V of activated MAP kinase
A solid support which directly binds an antigenic peptide containing a unique amino acid sequence between III and indirectly through a spacer and / or a high molecular weight carrier molecule which is not recognized by the first antibody (antibody of the present invention); ) Reagent containing the antibody of the present invention, (3) Reagent containing a labeled antibody against the antibody of the present invention, that is, a second antibody labeled with an enzyme or the like that recognizes and binds to the first antibody (antibody of the present invention) A reagent containing. Further, (4) an antigenic peptide containing a unique amino acid sequence which is an antigen or an active MAP
Kinase standardization solution, (5) buffer solution, (6) polypeptide that prevents non-specific adsorption and aggregate formation, additives such as surfactants, and (7) pipette, reaction vessel, calculation curve, etc. Good to include. The solid supports described above have a very wide range of designs and can have very different shapes depending on the particular purpose for which they are intended. For example, plates, spheres, plates, small rods, cells,
Examples include small bottles, small tubes, fibers and nets. As a specific example, a transparent plastic material such as a microtiter plate made of polyvinyl chloride or polystyrene, a small ball made of polystyrene and polystyrene latex, a tube or a rod can be used.

IV. Immunological staining of cells or tissues Active MA present in fixed cells or tissue specimens
This is a method for recognizing P kinase by the antibody of the present invention (first antibody) and detecting the antibody, which is useful for examining the abundance and localization in cells or tissues. The cells or tissues to be examined are fixed, and a specimen that can be observed with an optical microscope or an electron microscope is prepared. The reagent used for fixing cells or tissues is not particularly limited as long as it is suitable for observation under a microscope and does not lose the ability of the antibody of the present invention to recognize active MAP kinase as an antigen. Preferred are 3.7% formalin solution, 100% methanol, 100% ethanol, 4% paraformaldehyde solution, 1% glutaraldehyde solution and the like.
In order to recognize the active MAP kinase present in the fixed cell or tissue sample by the antibody of the present invention (first antibody), 0.1% (w / w
v) You may process with SDS. Then, by incubating the fixed cell or tissue sample for about 30 minutes to about 24 hours at 4 ° C. or room temperature, the activated MAP kinase present in the fixed cell or tissue sample is treated with the antibody of the present invention (first Antibody). Next,
A method for detecting the antibody will be described. The antibody of the present invention (first antibody) is an enzyme such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, etc. If labeled with
After incubation, the antibody of the present invention in a free state (first
The antibody of the present invention (first antibody) can be detected by removing the antibody) by washing, then allowing the substrate of the above-mentioned labeling enzyme to act, and measuring the reaction by color development or the like. For example, in the case of labeling with peroxidase, the combination of hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a color-developing reagent produces a brown or yellow color. When labeled with glucose oxidase, the substrate may be, for example, 2,2'-acid-di- (3-ethylbenzothiazoline-
6-sulfonic acid (ABTS) or the like is used. When a second antibody labeled with an enzyme or the like that recognizes and binds to the antibody of the present invention (first antibody) is used, after incubation,
The free antibody of the present invention (first antibody) is removed by washing and then incubated with the second antibody. Examples of the secondary antibody labeled with this enzyme or the like include peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, etc. An antibody to the first antibody (the antibody of the present invention) to which the enzyme of (1) is bound can be mentioned. As a specific example, when a rabbit antiserum is used as the first antibody (antibody of the present invention), as the second antibody, anti-rabbit immunoglobulin (IgG) and goat immunoglobulin (IgG) conjugated with peroxidase are preferred. be able to. The anti-rabbit IgG goat I
gG is commercially available and is easily available. For detection of the second antibody, the labeled antibody of the present invention (first
The same method as in the case of (antibody) can be mentioned. Hereinafter, the present invention will be described with reference to examples, but it goes without saying that the present invention is not limited to the following examples.

[0017]

【Example】

 Production Example 1 Preparation of Complex (Part 1: Unique Amino Acid Sequence
A process for forming a complete antigen (immunogen) of an antigenic peptide containing
Automatic amino acid synthesizer (apply
Polymerization by using 403A type manufactured by Dobio System Co., Ltd.
This produced 30 mg. The purity is HPLC
It was 95.5% by analysis. N- Asp-His-Thr-Gly-Phe-Leu-Thr (PO3H2) -Glu-Tyr (PO3H
2) -Val-Ala-C manufactured peptide (hereinafter referred to as the present peptide) 1
5.8 mg and glutaraldehyde 26.8 mg 2.
0 ml of 0.2M phosphate buffer (NaH₂PO_Four-Na ₂HP
O_Four, PH8.0), 25% glutaraldehyde 30 μl
Was slowly added with stirring, and the mixture was stirred at 10 ° C. overnight. Anti
After completion of the reaction, add an excess amount of sodium cyanoborohydride.
Reduced for 30 minutes. Next, add this mixture to a dialysis tube.
(Manufactured by Sanko Junyaku Co., Ltd.), at room temperature against 8M urea at 5 o'clock
For 10 hours at running temperature, 5 hours at room temperature and 5 hours at distilled water.
(During dialysis, exchanged with fresh distilled water 3 times). Dialysis
Then, the mixed solution is freeze-dried to obtain the KLH complex.
Got

Preparation Example 2 Preparation of Complex (Part 2: Linking of High Molecular Weight Carrier Molecule to Antigen Indirectly Attached to Solid Support) Bovine Serum Albumin (BSA) Instead of Keyhole Limpet Hemocyanin (KLH) A BSA complex is obtained in the same manner as in Production Example 2 except that is used.

Production Example 3 Step of immunizing mammals and step of obtaining antibody of the present invention The KLH complex obtained in Production Example 1 was dissolved in physiological saline to a concentration of 1 mg / ml. 2 ml of this aqueous solution
RAS [MPL (Monophosphoryl Lipid A) + TDM (Synthetic
Trehalose Dicorynomycolate) + CWS (Cell Wall Skeleto
n) Adjuvant system] (manufactured by Sigma)
Was added and mixed well. The obtained mixture was used as a New Zealand White rabbit (female, 14 weeks old, average 2.4 kg).
A liquid volume of 1 ml was administered per bird. The breakdown is 50 μl
Each was intradermally administered to 6 places on the back, 200 μl was intramuscularly administered to each hind leg, and 100 μl was subcutaneously administered to 3 places on the back. Then, after 4 weeks and 8 weeks, the administration was performed again in the same manner as the first administration. During this period, a small amount of blood was sampled from the rabbit ear vein and the antibody titer was measured. Since the antibody titer increased after the 3rd administration, 3 weeks after the 3rd administration,
100 μl of the above mixture was intravenously administered to the ear 4 times at intervals of 4 days. Five days after the final administration, whole blood was collected from the heart of the immunized test rabbit, the obtained blood was placed in a separapit tube (manufactured by Sekisui Chemical Co., Ltd.), incubated at 37 ° C. for 2 hours, and then centrifuged ( 3000rpm, 20min
, Room temperature) and the supernatant was collected to obtain antiserum. Furthermore, the obtained antiserum is treated at 56 ° C. for 30 minutes,
The complement system was inactivated. This was used as the (first) antibody in the following test.

Test Example 1 Analysis of Activated MAP Kinase by Immunoblotting Near-all MA of Xenopus laevis and almost all MAs in which almost all MAP kinases are activated
Using immature Xenopus laevis eggs in which P kinase is inactivated, and 4 types of cells derived from rat-derived 3Y1 cell line stimulated by epidermal growth factor and rat-derived 3Y1 cell line not given any stimulation Analysis of active MAP kinase was performed. The extract from unfertilized Xenopus laevis eggs was prepared by the following method. Gonadotropin (manufactured by Teikoku Organ Co., Ltd.) was injected into females of Xenopus laevis at a rate of 300 IU / animal, and the dose was 1 at 22 ° C.
After breeding for 2 hours, unfertilized eggs were taken out. 2% (w / v) cysteine hydrochloride (p
H7.8) and extracted with extraction buffer (composition: 60 mM β-glycerophosphate, 20 mM Tris-HCl (pH 7.5), 10 mM EGTA, 10 mM MgCl2,2
mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 2
0 μg / ml aprotinin) was added in an amount twice the volume of the unfertilized egg.
The mixture was homogenized while cooling with ice and centrifuged (200,000 xg, 20 min, 4 ° C), and the supernatant was collected as an unfertilized egg extract. Next, an extract from immature Xenopus laevis eggs was prepared by the following method. The ovaries were removed from females of Xenopus laevis and MBS-Ca ^{2 +} containing 1 mg / ml collagenase [composition: 88 mM NaCl, 1 mM KCl, 2.4 mM]
NaHC03, 0.82 mM MgSO4, 15 mM Tris-HCl (pH 7.6)] at 23 ° C.
Incubate for 2 hours, and then stage VI (immature egg) egg (the largest egg among the eggs, with the color of the plant pole side (cream) closest to the animal pole side (black) whitening)
Were selected with the naked eye, and the extraction buffer (composition: 60 mM β
-Glycerophosphoric acid, 20 mM Tris-HCl (pH 7.5), 10 mM EGTA, 10 mM M
gCl2, 2 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 20 μg / ml aprotinin) was added in an amount twice the volume of immature eggs. The mixture was homogenized while cooling with ice, centrifuged (200,000 xg, 20 min, 4 ° C), and the supernatant was collected as an immature egg extract. Next, an extract from the rat-derived 3Y1 cell line was prepared by the following method. Diameter 10
cm 3 Petri dish containing rat-derived 3Y1 cells containing 10% (v / v) fetal bovine serum in Dulbecco's MEM medium (Nissui Pharmaceutical Co., Ltd.)
The cells were cultivated at 37 ° C. to confluence and further cultured at 37 ° C. for 2 days in serum-free Dulbecco's MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) to synchronize the cells with the G0 / G1 phase. In this state, epidermal growth factor (EGF)
(Manufactured by Collaborative) was added at 30 nM, and the mixture was added at 37 ° C for 5
The stimulus was given for a minute to process. Epidermal growth factor (EG
Rat-derived 3Y1 cells stimulated by F) and rat-derived 3Y1 cells not given any stimulation were washed once with ice-cold HEPES buffer (pH 7.3).
0 μl extraction buffer [Composition: 20 mM β-glycerophosphate, 1
5mM Tris-HCl (pH7.7), 350mM Sucrose, 10mM metabisulfate, 5
mM KCl, 5 mM EGTA, 5 mM MgCl2, 0.1 mM EDTA, 6 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 20 μg / ml aprotinin] were added, and the cells were scraped with a rubber spatula and homogenized. Centrifuge the resulting homogenized solution (5000x
g, 5 min, 4 ° C), and further collect the supernatant by centrifugation (5
00,000xg, 20min, 4 ° C). After centrifugation, add 3Y to the supernatant
It was collected as a 1-cell extract. Prepared extract 100
MAP was separated from other proteins on the gel by electrophoresis (30 mA, 1 hour) using 10% (w / v) polyacrylamide gel (μg). IMMOBILO on this gel
Overlay N membrane (5x5cm, Millipore) and transfer buffer
[Composition: 24 mM Tris, 192 mM glycine, 0.1% (w / v) SDS + 20% (v / v)
Semi-transfer device (1mA / cm)
2, 90 minutes; MA using Biocraft, BE-300 type)
P-kinase and other proteins were transferred to the IMMOBILON membrane.
The obtained IMMOBILON membrane was then applied to a TBS solution containing Tween20 [composition: 0.05% (V / V) Tween20 + 20mM Tris-HCl (pH7.5), 500
After washing lightly with mM NaCl], a TBS solution containing skim milk (manufactured by Morinaga Milk Industry Co., Ltd.) for blocking [composition: 3
% (W / v) skim milk + 20mM Tris-HCl (pH7.5), 500mM NaC
1] for 1 hour at room temperature. After blocking, transfer the IMMOBILON membrane to a TBS solution containing Tween20 (composition: 0.1% (V / V) Tween20 + 20mM Tris-HCl (pH7.5), 500m
After lightly washing with M NaCl, a TBS solution containing BSA [composition: 1% (w / v) BSA +20 mM Tris-HCl (pH 7.5), 500 mM NaCl]
] The first antibody preparation diluted 200-fold with (Example 3
Immersed in 1 ml (prepared with the antiserum obtained in 1.) for 10 hours at 4 ° C. Tw the IMMOBILON membrane after the first antibody treatment
TBS solution containing een20 [Composition: 0.1% (V / V) Tween20 + 20mM
After washing with Tris-HCl (pH 7.5), 500 mM NaCl 3 times for 10 minutes each, a TBS solution containing skim milk (Morinaga Milk Industry Co., Ltd.) [composition: 3% (w / v) skim milk +20 mM Tris-HCl ( pH 7.
5), donkey anti-rabbit immunoglobulin (IgG, Amersham) solution (second antibody preparation solution) bound to peroxidase (HRP) diluted 5000 times with 500 mM NaCl] 2
Immerse in 0 ml for 30 minutes at room temperature. IM after treatment with secondary antibody
MOBILON membrane in TBS solution containing Tween 20 [Composition: 0.1%
V) Tween20 + 20mM Tris-HCl (pH7.5), 500mM NaCl] 15
After washing once for 5 minutes and 4 times for 5 minutes each, the first antibody remaining in the IMMOBILON membrane was detected by a color reaction using an ECL color development kit (manufactured by Amersham). As a result, when an extract prepared from an unfertilized Xenopus egg and an epidermal growth factor-stimulated rat-derived 3Y1 cell line was used as a sample, a color reaction due to the labeling of the second antibody was observed. When an extract prepared from immature Xenopus laevis eggs and the rat-derived 3Y1 cell line not given any stimulation was used as a sample, no color reaction due to the labeling with the second antibody was observed. From the above, the antibody of the present invention recognizes only active MAP kinase,
It was revealed that it showed substantially no cross-reactivity to inactive MAP kinase. Furthermore, it was also found that the antibody of the present invention similarly reacts against amphibian and mammalian origin active MAP kinases across animal species. The results are shown as a schematic diagram (see FIG. 1). In addition, there is one type of MAP kinase in Xenopus laevis, and
Although there are two kinds of kinases, one band was observed in lane B and two bands (active MAP kinase) were observed in lane D. The results are shown as a schematic diagram (see FIG. 1). Furthermore, with respect to the antibody of the present invention obtained in Example 3, the degree of cross-reactivity to inactive MAP kinase was examined. The degree of cross-reactivity depends on the activated MA
When the substrate protein concentration at which P-kinase can be detected by the immunoblotting method is 1, and the concentration of the substrate protein at which inactive MAP kinase is detected by the method is C, it is 1 / C. Was decided. As a result, the antibody of the present invention obtained in Example 3 was
Selective recognition of less than 100 was possible. The results are shown as a schematic diagram (see FIG. 2).

Test Example 2 Analysis of Active MAP Kinase by Immunoprecipitation Method Using the Antibody of the Present Invention Almost all Xenopus laevis eggs in which almost all MAP kinases are activated or conversely almost all MA
An extract from immature Xenopus laevis eggs in which P kinase was inactivated was prepared by the same method as in Test Example 1. 40 μl of the prepared extract, the first antibody (Example 3
5 μl of the antiserum obtained in 1., 5 μl of protein A Sepharose 6 MB solution (Pharmacia Fine Chem.) And 5.5 μl of 1% (w / v) SDS aqueous solution were mixed, and the mixture was mixed at 4 ° C. for 1.5 minutes. Incubated for hours. Tween solution containing Tween40 in the mixture after treatment [Composition: 0.1% (V / V) Twee
1 ml of n40 + 20 mM Tris-HCl (pH 7.5), 500 mM NaCl] was added and mixed, and the precipitate was recovered by centrifugation (5000 rpm, 2 min, 4 ° C.). TBS solution containing Tween40 with the recovered precipitate as the final concentration [Composition: 0.1% (V / V) Tween40 + 20 mM Tris-HC
1 (pH 7.5), 500 mM NaCl] 1 ml was washed twice. Load the washed pellet with loading buffer (Tris-SDS-BME Separ
10 μl of asol, manufactured by EnproTech Co., Ltd., Daiichi Pure Chemical Co., Ltd. was added, and this was heat-treated (90 ° C., 3 minutes) to release the active MAP kinase immunoprecipitated together with the first antibody. In this way, an operation of separating and extracting only active MAP kinase was performed. 10 μl of each of the solutions obtained by the above operation or the extract prepared without the above operation was added to myelin basic protein.
MAP kinase and other proteins were separated on the gel by electrophoresis (30 mV, 1 hour) using a 10% (w / v) polyacrylamide gel containing 0.5 mg / ml. 20% (w / v) 2-propanol / 50 mM Tris-HCl (pH
8.0) Shake while soaking in 50 ml of solution for 1 hour at room temperature (change wash solution once every 30 minutes). Then, for washing, it was shaken while being immersed in 50 ml of A buffer [composition: 5 mM mercaptoethanol / 50 mM Tris-HCl (pH 8.0)] for 1 hour at room temperature (the washing solution was changed once every 30 minutes). Next, the washed gel was shaken in 50 ml of A buffer containing 6M guanidine hydrochloride at room temperature for 1 hour, and then left still in 50 ml of A buffer containing 0.04% (V / V) Tween 40 at 4 ° C. for 14 hours ( On the way, change to a new cleaning solution 4 times). Finally, B buffer [composition: 0.1 mM EGTA [ethylene glycol bis (β-aminoethyl ether) -N,
N, N ', N'-tetraacetic acid], 15 mM MgCl2, 2 mM DTT (dithiothreitol), 40 mM HEPES [N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid] -NaOH
(pH 8.0)] Shaking while soaking in 50 ml at room temperature for 30 minutes. The gel thus treated was treated with 25 μM ATP,
B buffer 15 containing 25 μCi [γ- 32 P] ATP
After soaking in 1 ml at room temperature for 1 hour, wash thoroughly with 250 ml of 5% (v / v) trichloroacetic acid / 1% (v / v) sodium pyrophosphate solution overnight (change to new washing solution 4 times on the way) ). Next, the amount of phosphorylation of myelin basic protein contained in the gel prepared as described above was measured by an image analyzer (Fujix BAS2000 manufactured by Fuji Photo Film Co., Ltd.), and the amount of active MAP kinase was quantified from this measured value. did. As a result, when the extract from unfertilized Xenopus laevis eggs or immature eggs was directly electrophoresed,
Phosphorylation of myelin basic protein by proteins other than MAP kinase was observed. On the other hand, when the solution obtained by immunoprecipitation of the extract from unfertilized Xenopus laevis eggs using the antibody of the present invention was electrophoresed, phosphorylation of myelin basic protein by proteins other than MAP kinase was not observed. In addition, when a solution obtained by immunoprecipitating an extract from immature Xenopus laevis eggs using the antibody of the present invention was electrophoresed, phosphorylation of myelin basic protein was not observed (see FIG. 3).

Furthermore, a solution prepared by immunoprecipitating an extract from unfertilized or immature Xenopus laevis eggs prepared as described above with the antibody of the present invention is used as a sample, and the "first antibody preparation ( Prepared using the antiserum obtained in Example 3) "instead of" active and inactive M
The active MAP kinase was analyzed according to the immunoblotting method described in Test Example 1 using "antiserum that reacts with both AP kinases". As a result, when a solution obtained by immunoprecipitating an extract from an unfertilized egg with the antibody of the present invention was used as a sample, a color reaction due to the labeling of the second antibody was observed,
On the other hand, when a solution obtained by immunoprecipitating an extract from an immature egg with the antibody of the present invention was used as a sample, no color reaction due to the labeling of the second antibody was observed. Therefore,
It was also confirmed that the antibody of the present invention recognizes only active MAP kinase and specifically separates active MAP kinase (by immunoprecipitation).

Test Example 3 Activated M by indirect competitive inhibition method
Analysis of AP Kinase (1) Using a polystyrene 96-well microtiter plate,
The BSA complex obtained in Example 2 was 1 to 10 μg /
coating solution containing 14 ml of sodium chloride
10 mM phosphate buffer (NaH containing 0 mg / l)₂
PO_Four-Na₂HPO_Four, PH 7.4)] 0.1 ml / well
And then incubate overnight at 4 ° C.
After removing the coating solution, the BSA composite was used.
10 coated microtiter plates
mM phosphate buffer (NaH₂PO _Four-Na₂HPO_Four,
It was washed once by immersing it in water (pH 7.4), and the mixture was washed at
Paper towel with the top of the crotiter plate facing down
Remove water from the plate by tapping lightly on top
To do. Then, add 1% (w / v) to the microtiter plate.
Si serum albumin was added at a rate of 0.1 ml / well
Then, incubate at room temperature for 1 hour. And again 4 ° C
Microtiter plates stored at 0.05% (w / v)
 10 mM phosphate buffer containing Tween 20 (NaH₂PO
_Four-Na₂HPO_Four, PH 7.4) and washed 3 times.
Next, the first antibody obtained in Production Example 3 was added at 0.5 mg /
10 mM phosphate buffer containing bovine serum albumin at a concentration of ml
Impulse liquid (NaH₂PO_Four-Na₂HPO_Four, PH 7.4) 1
0 to 10⁸A 1-fold diluted 1st antibody preparation was mixed with the above
Addition of 0.1 ml / well to the rotiter plate
Then, incubate at room temperature for 2 hours. And the first antibody
After removing the preparation, it contains 0.05% (w / v) of Tween20
10 mM phosphate buffer (NaH₂PO_Four-Na₂HPO
_Four, PH 7.4) and washed 3 times. Next, commercially available Pell
Oxidase labeled anti-rabbit immunoglobulin (IgG)
Gui immunoglobulin (IgG) [Seikagaku Corporation]
10 mM containing bovine serum albumin at a concentration of 0.5 mg / ml
Phosphate buffer (NaH₂PO_Four-Na₂HPO_Four, PH
The second antibody preparation diluted 5000 times with 7.4) was mixed with the above solution.
Add 0.1 ml / well to the crotiter plate
Then, incubate at room temperature for 2 hours. And the second
After removing the antibody preparation, add 0.05% (w / v) Tween20.
10 mM phosphate buffer containing (NaH₂PO_Four-Na₂H
PO_Four, PH 7.4) and washed three times at 4 ℃.
Rotate plate top side down on paper towel
Remove water from the plate by tapping lightly with
It Then, at a concentration of 0.32 μg / ml, o-phenylenediamine
And 0.5M containing hydrogen peroxide at a concentration of 0.01% (v / v)
Of sodium citrate solution prepared immediately before use,
Drug and 0.15M phosphate buffer (NaH₂PO_Four-Na
₂HPO_Four, PH 6.0) to the above microtiter plate
0.1 ml / well to each of the
Cover the titer plate with aluminum wheels and use 3 at room temperature.
Incubate for 0 minutes. And 0.05 ml of 4N sulfuric acid
The enzyme reaction is stopped by adding
ー Multi-scanning spectrophotometry
Using a photometer (manufactured by Hitachi, Ltd.) at 492 nm
The absorbance of is measured.

Test Example 4 Active M by Indirect Competitive Inhibition Method
Analysis of AP Kinase (Part 2) The primary antibody obtained in Production Example 3 was treated with 10 mM phosphate buffer (NaH ₂ PO ₄ —Na ₂ HPO ₄ , pH 7.) containing bovine serum albumin at a concentration of 10 mg / ml. Prepare a 10-fold dilution series in 4). On the other hand, the KLH complex obtained in Production Example 1 was treated with 10 mM phosphate buffer (NaH ₂ PO ₄ -Na) containing bovine serum albumin at a concentration of 10 mg / ml.
Prepare a corresponding 10-fold dilution series with ₂ HPO ₄ , pH 7.4 (standardized solution). The former first antibody preparation and the latter K
Mix LH complex preparation (standardization solution) and incubate overnight at about 20 ° C. Next, the above reaction solution was added to the microtiter plate coated with the BSA complex at a rate of 0.1 ml / well, and then at 20 ° C. for 1 hour.
Incubate for hours. And after removing the reaction solution,
10mM phosphate buffer containing Tween20 of 0.05% (v / v) ( NaH 2 PO 4 -Na 2 HPO 4, pH7.4) and washed 3 times with. The following operations (for example, reaction with the second antibody, color reaction with a labeling enzyme, etc.) are performed in the same manner as in Test Example 3 to prepare a calibration curve for quantification of the KLH complex.

[0025]

EFFECTS OF THE INVENTION By using the antibody of the present invention, it has become possible to analyze active MAP kinase with high accuracy, which can be processed simply and rapidly.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the results of analysis of active MAP kinase by immunoblotting. Lane A is an immature Xenopus laevis egg, Lane B is an unfertilized Xenopus laevis egg, Lane C is a rat-derived 3Y1 cell line that has not been given any stimulation, and Lane D is a rat-derived 3Y1 cell line stimulated by epidermal growth factor. Was used as a sample.
In Lanes B and D, the color reaction due to the labeling of the second antibody was observed, while in Lanes A and C, the color reaction due to the labeling of the second antibody was not observed at all. Also, there is one type of Xenopus MAP kinase and two types of rat MAP kinase, but one band in lane B and two bands in lane D (active MAP).
Kinase) was observed in each case.

FIG. 2 is a schematic diagram showing the results of examining the degree of cross-reactivity of the antibody of the present invention with inactive MAP kinase. Lanes A and B are extracts from unfertilized Xenopus laevis eggs (1/100, 1/25 amounts, respectively), and Lanes C, D, E, and F are immature Xenopus laevis eggs (1/100, 1/25, respectively). (1/5, 1 volume). In lanes A and B, MAP
The kinase band was specifically observed, while the MAP kinase band was not specifically observed in lanes C, D, E and F.

FIG. 3 is a diagram showing an analysis result (immunoassay method) of active MAP kinase by an immunoprecipitation method using the antibody of the present invention. Lane A is the case where the extract from unfertilized Xenopus laevis eggs was electrophoresed as it is. Lanes B to F are the cases where the solution obtained by immunoprecipitating the extract from unfertilized Xenopus laevis eggs using the antibody of the present invention was subjected to electrophoresis. In Lane A, phosphorylation of myelin basic protein by MAP kinase and other proteins was observed. On the other hand, in lanes BF, MA
No phosphorylation of myelin basic protein by proteins other than P kinase was observed.

Claims (4)

[Claims] 1. A subdomain VI of active MAP kinase.
An antibody which can be obtained by using a complex in which an antigenic peptide containing a unique amino acid sequence between I and subdomain VIII is directly or indirectly bound to a high-molecular-weight carrier molecule through a spacer as an antigen (1 ) The unique amino acid sequence is N- Thr (PO3H2) -Glu-Tyr (PO3H2) -C, (2)
Reacts with active MAP kinase in the presence of 0.1% (w / v) SDS and does not substantially react in the absence of (3) cross-reactivity to inactive MAP kinase An antibody that recognizes active MAP kinase, characterized in that it is not shown. 2. Subdomain VI of active MAP kinase
After immunizing a mammal against a complex in which an antigenic peptide containing a unique amino acid sequence between I and subdomain VIII is directly or indirectly bound to a high molecular weight carrier molecule, the mammal is immunized. The method for producing the antibody according to claim 1, wherein blood is collected from the blood, and the blood is separated and purified from the blood. 3. A method for immunologically detecting active MAP kinase, which comprises detecting the antibody according to claim 1, which has been made to act on active MAP kinase, or an antibody against the antibody, by a label of the antibody. 4. Subdomain VI of active MAP kinase
A solid support in which an antigenic peptide containing a unique amino acid sequence between I and subdomain VIII is directly or indirectly bound via a spacer and / or a high molecular weight carrier molecule which is not recognized by the antibody of claim 1. A kit for immunological detection and analysis of active MAP kinase, which comprises a material, a reagent containing the antibody according to claim 1, and a reagent containing a labeled antibody against the antibody according to claim 1. .