JP2001343389A - Inspection method of cancer by measurement of autoantibody against mdm2, and its reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new inspection method of cancer. SOLUTION: In this inspection method, an autoantibody against MDM2 in blood is measured by an immunological method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring the presence of M present in a sample.
A method for testing cancer by measuring autoantibodies to DM2,
It relates to the reagent and the kit.

[0002]

2. Description of the Related Art MDM2 (Murine Double)
minute-2) is a gene product localized in the cell nucleus like histone, p53, hormone receptor, etc.
It is known to be frequently amplified in sarcomas.
It has been reported that the autoantibody exists in various gynecological patients (Cancer Letter).
s, 96, 111-115 (1995)).

[0003] It is generally known that certain gene products localized in the cell nucleus are highly expressed as cells become cancerous as compared with normal cells. On the other hand, in a tumor-bearing host, it is known that tumor cells are destroyed by a tumor immune reaction, and a gene product present in the cell nucleus is transferred out of the cell. As a result, production of humoral antibodies is known. Autoantibodies against histone, p53, etc. are such examples (JP-A-4-3276).
No. 6, JP-A-7-505719 (International Publication W
O93 / 21529) and JP-A-9-229933.
Gazette (British Patent No. 2241782).

[0004] However, the conventional measurement of autoantibodies has a problem that cancer specificity is low and it is difficult to distinguish between cancer and non-cancer.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a simpler method for testing cancer using autoantibodies, particularly a test method capable of diagnosing early cancer, a reagent therefor, and a kit therefor.

[0006]

Means for Solving the Problems As a result of various studies to achieve the above object, the present inventors have found that the measurement of autoantibodies against MDM2 in the blood of various cancer patients is effective for cancer diagnosis. And completed the present invention.

That is, the present invention provides (1) a method for testing a cancer, which comprises measuring an autoantibody against MDM2 present in a sample. (2) The method according to the above (1), wherein the cancer is early cancer. Test method (3) The test method according to (1), wherein the cancer is epithelial cancer (4) The test method (3), wherein the epithelial cancer is lung squamous cell carcinoma, lung adenocarcinoma or small cell lung cancer (5) The test method according to any one of (1) to (4), wherein the measurement of the autoantibody is performed by an immunological measurement method.

(6) The test method according to the above (5), wherein the immunological assay is a competitive assay, an inhibition assay or a sandwich assay. (7) The antigen used in the immunological assay is MDM2. (5) or the test method according to (6) above, which is a partial peptide of (8) the test method according to (5) above, which uses an anti-MDM2 antibody or a label thereof (9) the anti-MDM2 antibody is a part of MDM2 (8) an antibody obtained by using a peptide as an antigen or a modified product thereof;
(10) The test method according to (7) above, wherein the partial peptide of MDM2 is a partial peptide of the N-terminal region of MDM2 or a modified product thereof.

(11) The above (9) or (1), wherein the partial peptide of MDM2 is immobilized on a solid phase.
(12) a peptide shown in the following (a) or (b): (a) a function having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and binding to an MDM2 autoantibody; (B) a peptide having an amino acid sequence in which some amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, and having a function of binding to an MDM2 autoantibody (13) A cancer test reagent containing an anti-MDM2 antibody or a labeled product thereof (14) MDM2, a partial peptide thereof, or a cancer test reagent containing a labeled product thereof (15) MDM2 partial peptide Is a partial peptide of the N-terminal region of MDM2 or a modified product thereof (14).
Reagents for cancer testing described in

(16) The cancer test reagent according to the above (14) or (15), wherein the partial peptide of MDM2 is immobilized on a solid phase. (17) The partial peptide of MDM2 is as follows: Peptide shown in a) or (b): (a) Peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and having a function of binding to an MDM2 autoantibody (b) SEQ ID NO: 1 in the sequence listing (14) to (16), which are peptides having an amino acid sequence in which some amino acids are deleted, substituted or added in the amino acid sequence shown in (1) and having a function of binding to an MDM2 autoantibody.
(18) The cancer test reagent according to any one of (13) to (1), wherein the cancer is epithelial cancer.
7) A cancer test reagent according to any one of the above (19). (19) A cancer test reagent comprising the cancer test reagent according to any one of the above (13) to (18). Kit (20) The present invention relates to the kit for cancer testing according to the above (19), comprising a standard solution for preparing a standard curve.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for detecting MD present in a sample.
This is a cancer test method by measuring autoantibodies against M2. The type of cancer to be treated may be epithelial cancer or non-epithelial cancer, but is preferably epithelial cancer such as gastrointestinal cancer (stomach cancer, esophagus cancer, colon cancer, liver cancer, pancreatic cancer, Gallbladder cancer), lung cancer and breast cancer. The sample used in the present invention is not particularly limited as long as it is a body fluid containing the autoantibody, but preferably includes blood, serum, plasma and the like, and more preferably human blood, serum and plasma.

MDM2 is a gene product localized in cells, and the transcriptional activity of that gene is enhanced by p53, one of the tumor suppressor gene products. It is also known to bind to p53 and suppress the transcription activity of p53.
In recent years, regarding the mechanism of suppressing the action of p53 by MDM2,
It was found that MDM2 acts as a ubiquitin ligase, ubiquitinates p53 and leads to degradation. Therefore, M
DM2 is highly expressed in various cancer cells, and p5
By acting as a decomposing enzyme of No. 3, it is thought to play an important role in the mechanism of cancer development.

An autoantibody against MDM2 is an antibody produced by autoantibody-producing cells against MDM2 existing in a living body and has a property of specifically binding to MDM2.

The method for measuring autoantibodies to MDM2 in blood is not particularly limited, but an immunological measurement method is suitable. For example, there is a method using an antibody obtained by immunizing an animal with MDM2 or a partial peptide of MDM2 (hereinafter, simply referred to as MDM2 partial peptide) as an antigen. A method using the MDM2 partial peptide as an antigen is as follows.
As described later, it is more preferable in terms of stability of the quality of the antigen, availability, and the like. The animal species to be immunized to obtain the antibody is not limited, but preferably includes rabbits, goats, mice, rats, horses, pigs, cows, chickens and the like. The antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.

In the present invention, unless otherwise specified, an anti-MDM2 antibody refers to an antibody obtained using MDM2 as an antigen and / or an antibody obtained using MDM2 partial peptide as an antigen. In some cases, the term “anti-MDM2 antibody” is used to include a modified form that does not belong to a labeled form, such as a biotinylated antibody. These antibodies may optionally be used as labeled antibodies. Preferred examples of the anti-MDM2 antibody include an antibody obtained by using the MDM2 partial peptide as an antigen or a modified product thereof (such as a biotinylated anti-MDM2 antibody).

As a measurement method using an antibody, for example, a competition method, an inhibition method, a sandwich method and the like can be mentioned. The MDM2 or the MDM2 partial peptide used in the measurement in the present invention also includes those modified products in which a part of the amino acids constituting them is modified by biotinylation or the like. The competitive method is based on the above-described anti-MD labeled with a labeling substance.
The M2 antibody and the autoantibody in the sample were compared with MDM2 or MDM.
The two partial peptides are allowed to react competitively, and the amount of the above-mentioned anti-MDM2 antibody bound thereto is measured. From the value, the amount of autoantibody in the sample is determined. Further, the unlabeled anti-MDM2 antibody and the autoantibody in the sample were compared with MDM2 or MDM2.
Competitively reacts with the DM2 partial peptide, and then reacts with an antibody against the above-mentioned anti-MDM2 antibody, which is labeled with a labeling substance, or with avidin or avidin analog or a derivative thereof labeled with a labeling substance. Then, the amount of the anti-MDM2 antibody bound to MDM2 or MDM2 partial peptide may be measured using the label.

The inhibition method comprises reacting an autoantibody in a sample with MDM2 or an MDM2 partial peptide and then reacting the anti-M
This anti-MDM2 against MDM2 was reacted with a DM2 antibody.
The amount that inhibits antibody binding is measured, and the amount of autoantibody in the sample is determined from the value. The above anti-MDM used at this time
2 antibody may be labeled with a labeling substance. Alternatively, after reacting the anti-MDM2 antibody, an antibody against the anti-MDM2 antibody, which is labeled with a labeling substance, is reacted, and the anti-MDM2 antibody is reacted. The amount that inhibits antibody binding may be measured.

In the sandwich method, when the autoantibody is a bivalent antibody having two sites that react with the antigen, the autoantibody in the sample is immobilized on a solid-phased MDM2 or the same MDM2 partial peptide, and labeled with a labeling substance. MDM2, labeled MDM2
An antibody against a partial peptide or an autoantibody labeled with a labeling substance is sandwiched and detected, and the amount of the autoantibody in the sample is determined from the value. Since the sample contains a large amount of antibodies other than autoantibodies against MDM2,
It non-specifically adsorbs and binds to the solid phase. Therefore, when a human-derived specimen is measured using a labeled antibody, it is preferable to use a labeled antibody other than an anti-human antibody in order to obtain a more accurate measurement value.

As the labeling substance, for example, radioactive substances such as ¹²⁵ I and ³ H, enzymes such as peroxidase, β-galactosidase and alkaline phosphatase, fluorescent substances and luminescent substances can be used. The labeling method is appropriately selected depending on the labeling substance and the substance to be labeled. These labels are
It may be directly bonded to the substance to be labeled, or may be indirectly bonded using, for example, a biotin-avidin reaction.

Next, the antigen used in the immunoassay according to the present invention will be described. MDM2 used as an antigen in the present invention may be one extracted and purified from tissues, one produced artificially by genetic recombination, chemical synthesis, or the like, or one modified within a range that does not lose the function of MDM2 binding to an autoantibody. Either may be used. Further, the MDM2 partial peptide used as an antigen in the present invention may be any part of the amino acid sequence of MDM2 as long as it has a function of binding to an MDM2 autoantibody,
The size of the peptide is not particularly limited as long as it is an MDM2 partial peptide, and may be an MDM2 partial peptide modified within a range that does not lose the function of MDM2 binding to an autoantibody. The MDM2 partial peptide may be appropriately cut out from MDM2 with an enzyme or the like, or may be artificially produced by genetic recombination, chemical synthesis, or the like as in the case of MDM2. The MD
The M2 partial peptide preferably has about 5 to 30 amino acids, more preferably about 10 to 20 amino acids in terms of antigen performance and ease of use.

The modified MDM2 or modified MDM2 partial peptide described above includes MD
Some amino acids in the amino acid sequence of MDM2 have been substituted with other amino acids or some amino acids have been deleted, and some amino acids have been added to the extent that the binding function of M2 to autoantibodies is not significantly inhibited. And the like. In addition, remarkably inhibiting the binding function of MDM2 to an autoantibody means a case where the binding function to an autoantibody is low and the object of the present invention is not achieved. In view of availability, stability of quality, and ease of construction of a measurement system, a partial peptide is preferable, and a chemically synthesized partial peptide is usually more preferable. The ease of obtaining the partial peptide, particularly the partial peptide by chemical synthesis, includes, for example, the fact that complicated operations for protein expression and extraction and purification from tissues are not required. The stability of the quality includes that a product of constant purity can be obtained at any time. Such a partial peptide is, for example, 10 to 10 amino acids from the known amino acid sequence of MDM2.
Approximately 20 residues are selected, and if necessary, some amino acids are deleted or substituted with other amino acids or one to several amino acids are added. As a MDM2 partial peptide, chemical synthesis such as Fmoc method is performed. Can be manufactured. As a preferred example, the number of amino acids in the N-terminal region of MDM2 is 1
Those having 0 to 20 residues, more specifically, partial peptides from the 3rd to the 22nd residue on the N-terminal side (MDM2 / N described later) described in the above (a) can be mentioned.

The peptide (a) of the present invention is an MDM
A 2N-terminal region, specifically, a partial peptide (MDM2 / N) described below from the 3rd to 22nd residues on the N-terminal side;
(B) is an MDM2 / N partial peptide modified within a range that does not lose the function of MDM2 binding to an autoantibody. Specifically, the peptide of (a) has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and the peptide of (b) has some of the amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. It has a deleted, substituted or added amino acid sequence and has a function of binding to an MDM2 autoantibody. The peptide (a) is a region having a relatively high function of binding to the MDM2 autoantibody in the amino acid sequence of MDM2, and is a region expected to have high hydrophilicity.

When an autoantibody against MDM2 in the sample of the present invention is measured by an immunoassay, it can be measured by immobilizing MDM2 or its partial peptide as an antigen on a solid phase. As a method for immobilization, a known protein immobilization method can be used as it is. For example, physical adsorption to a solid phase such as a microplate, polystyrene beads, magnetic fine particles, or the like, MDM2 or a partial peptide thereof can be covalently bonded using a cross-linking agent, or biotin-avidin bond can be used. If necessary, a functional group or a linker may be introduced to strengthen the bond with the solid phase, or the solid form may be bound to the solid phase in a preferable configuration for the antigen-antibody reaction so that the reactivity with the antibody is not impaired.

The cancer test reagent of the present invention contains MDM2, a partial peptide thereof or a label thereof, and if necessary, an anti-MDM2 antibody or a label thereof. The cancer to be tested is preferably the above-mentioned epithelial cancer. Examples of the MDM2 partial peptide contained in the cancer test reagent of the present invention include the same peptides as the aforementioned MDM2 partial peptide. Preferred is a partial peptide at the N-terminal part of MDM2, and specifically has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which some amino acids are deleted, substituted or added. Peptides. Further, the MDM2 partial peptide may be immobilized on a solid phase as described above. Anti-MDM2
Examples of the antibody or its label include the above-mentioned anti-MDM2 antibody or its label. In addition, a qualitative reagent for determining the presence or absence of cancer based on a signal such as coloring when an autoantibody having a concentration equal to or higher than or less than a certain concentration is obtained is also included in the present invention.

The cancer test kit of the present invention is a kit containing the above-mentioned cancer test reagent, and is a kit containing a cancer test reagent containing MDM2, a partial peptide thereof or a label thereof. . Examples of the reagent for cancer test containing MDM2, its partial peptide, or a labeled product thereof include those described above. Kits can also be used for competition, inhibition,
Although it depends on the measurement principle, such as the sandwich method, MDM2
A standard solution for preparing a standard curve for the concentration of autoantibodies to the sample, a diluted solution of the sample, a substance to which a colored substance such as a radioisotope, an enzyme, a fluorescent substance, gold colloid, etc. It may be a configuration that includes. In the case of a kit for detecting an immune reaction using an enzyme, for example, a substrate solution and an enzyme reaction stop solution, B / F (Bind and Fre
e) When a solid phase requiring separation is used, it may contain a washing solution or the like.

The kit of the present invention selects and uses necessary components from these configurations, and may further include components included in the configuration of a general test kit. If the reagents that make up these kits are stable for storage, they can be incorporated into the kit in their original form.If they are unstable, they can be reconstituted or diluted, for example, in the form of a lyophilized product or a concentrated solution. May be included with the solution. More specifically, for example, using a 96-well microplate on which MDM2 is immobilized, an autoantibody against MDM2 contained in the test sample (hereinafter sometimes referred to as MDM2 autoantibody for convenience) is used for the immobilized antigen. In the case of a kit for quantifying MDM2 autoantibodies contained in a test sample by competing with an anti-MDM2 antibody prepared by immunizing a second animal different from the test sample, for example,
(1) a microplate on which MDM2 or MDM2 partial peptide is immobilized; and (2) an anti-MDM2 prepared as a standard solution by immunizing the same animal species as the test sample or a third animal species different from the competitive anti-MDM2 antibody. Several different concentrations of anti-MDM2 antibody solution prepared from the antibody, (3)
A buffer for diluting the test sample, (4) an enzyme-labeled anti-immunoglobulin antibody specific to the second animal species in which an anti-MDM2 antibody that competes with an autoantibody to MDM2 contained in the test sample was prepared, 5) a coloring solution containing a dye or the like serving as a substrate for the enzyme, (6) a stop solution for stopping the enzyme reaction,
(7) Consisting of necessary reagents selected from a washing solution or the like for washing the microplate between each reaction step.

The kit of the present invention contains MDM in a test sample.
In addition to the quantification kit for measuring the concentration of the autoantibody to No. 2, a qualitative kit for determining that an autoantibody at a certain concentration or more or at a certain concentration or less exists in the test sample is also included. Such an example includes an autoantibody at a certain concentration or higher, such as an immunochromatographic test paper using MDM2 immobilized on a nitrocellulose membrane and an antibody labeled with colloidal gold against immunoglobulin contained in a test sample. In a test sample to be tested, a coloring line appears at the determination site, and in a test sample having a concentration lower than that, no coloring is observed, so that a kit for determining the presence or absence of cancer can be mentioned.

Next, one of the methods of the present invention for testing for cancer using the autoantibody against MDM2 as an indicator of the present invention will be described in more detail step by step.

The reactivity between the MDM2 partial peptide antigen and the antibody thereto can be confirmed, for example, by the following procedure. 1) The synthetic MDM2 partial peptide antigen is immobilized on a microtiter plate by physical adsorption. 2) First reaction A rabbit polyclonal anti-MDM2 antibody (IgG) prepared by immunizing a rabbit with the MDM2 partial peptide is added to each well of the plate on which the MDM2 partial peptide antigen is immobilized, and reacted. 3) Second reaction After removing the solution in the wells by suction, each well is washed, and an enzyme-labeled anti-rabbit IgG antibody solution is added and reacted. 4) Third reaction After the solution in the wells is removed by suction, each well is washed, and a color former and a substrate are added and reacted. 5) Stopping the reaction Stop the color reaction by adding a reaction stop solution. 6) Measurement of Absorbance The absorbance of each well is measured to confirm the adsorption of the partial peptide antigen to the plate and to confirm that an antibody specifically reacting with the antigen is formed.

The partial peptide antigen can be selected, for example, by the following procedure. 1) Each MDM2 partial peptide antigen is immobilized on a microtiter plate by physical adsorption. 2) First reaction To each well of the MDM2 partial peptide antigen-immobilized plate is added a patient serum which is considered to have a high autoantibody titer against MDM2 and reacted. 3) Second reaction After removing the solution in the wells by suction, each well was washed, and a certain amount of a rabbit polyclonal anti-MDM2 antibody (IgG) solution against the same MDM2 partial peptide antigen used for immobilization was added and reacted. Let it. 4) Third reaction After the solution in the well is removed by suction, each well is washed, and an enzyme-labeled anti-rabbit IgG antibody solution is added and reacted. 5) Fourth reaction After the solution in the well is removed by suction, each well is washed, and a coloring agent and a substrate are added and reacted. 6) Stopping the reaction Stop the color reaction by adding a reaction stop solution. 7) Measurement of absorbance Measure the absorbance of each well. 8) Selection of partial peptide antigen In the above case, the reactivity of the MDM2 autoantibody in the test serum to each MDM2 partial peptide antigen is reflected in the absorbance. That is, the higher the reactivity of the partial peptide antigen, the more the amount of MDM2 autoantibody bound in the serum increases, and as a result,
The amount of the partial peptide antigen that reacts with the anti-MDM2 antibody decreases, and the amount of the anti-MDM2 antibody that binds to the immobilized MDM2 antigen decreases. Therefore, an enzyme-labeled anti-rabbit IgG was added to the bound anti-MDM2 antibody (rabbit polyclonal antibody).
Due to the decrease in the amount of antibody binding, color development is weakened and the absorbance is reduced. From the above, it is considered that the lower the absorbance is, the higher the reactivity of the antigen is. Therefore, the antigen having the lowest absorbance is selected from each MDM2 partial peptide antigen.

The measurement of the anti-MDM2 autoantibody by the inhibition method can be performed, for example, by the following procedure. 1) The MDM2 partial peptide antigen is immobilized on a microtiter plate by physical adsorption. 2) First reaction The test serum or buffer alone is added to each well of the plate on which the MDM2 partial peptide antigen-immobilized plate is reacted. 3) Second reaction After aspirating and removing the solution in the wells, each well was washed, and a rabbit polyclonal antibody (IgG) solution against the MDM2 partial peptide was added to the wells reacted with the test serum to cause a reaction. Rabbit polyclonal antibody standard solution is added to the reacted wells for reaction. The antibody standard solution was a rabbit polyclonal anti-MDM2 antibody (IgG) against the MDM2 partial peptide added to the well in which the test serum was reacted.
Is a concentration series. 4) Third reaction After the solution in the well is removed by suction, each well is washed, and an enzyme-labeled anti-rabbit IgG antibody solution is added and reacted.

5) Fourth Reaction After the solution in the wells is removed by suction, each well is washed, and a color former and a substrate are added and reacted. 6) Stopping the reaction Stop the color reaction by adding a reaction stop solution. 7) Measurement of absorbance Measure the absorbance of each well. 8) Determination of MDM2 autoantibody concentration in blood A standard curve was drawn from the absorbance of the well to which the rabbit polyclonal anti-MDM2 antibody standard solution was added, and the absorbance of the well to which the test serum was added was fitted to the standard curve. The concentration of rabbit polyclonal antibody that reacted despite being inhibited by binding is determined. The difference between the added rabbit polyclonal anti-MDM2 antibody concentration and the measured concentration is defined as the autoantibody concentration against blood MDM2 (blood MDM2 autoantibody concentration).

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

【Example】

Embodiment 1 Preparation of MDM2 partial peptide and selection of antigen with high cancer specificity Synthesis of various MDM2 partial peptides From the amino acid sequence of MDM2, the MDM2 partial peptide selects, from the N-terminal region, the C-terminal region, and the intermediate region, a region having high hydrophilicity and a region expected to be an antigenic determinant, Three types of MDM2 with 14 to 20 residues
The partial peptide was chemically synthesized by the Fmoc method. MDM2 / N for the N-terminal region, MDM2 / N for the C-terminal region
C, the one in the middle area is MDM2 / NC.

2. Preparation of Microtiter Plate with MDM2 Antigen Immobilized Microtiter plate (AquaBin) was prepared by mixing the above three kinds of synthetic MDM2 partial peptides with 0.1 M carbonate buffer (pH 9.0) at 5 μg / 150 μl / well.
d, made by Danish M & E). Room temperature 3
After reacting for an hour, the antigen solution in each well is removed by suction, and 1 ml of 0.1 M carbonate buffer (pH 9.30) is used using a microplate washer (Minilab Washer, manufactured by Lifetec).
Wash each well twice in 0). Then, pH7 containing 15% polyethylene glycol (MW4000), 10 mM diethanolamine and 0.1% sodium azide.
25 of 0.05M phosphate buffer (PBS)
Add at 0 μl / well and seal tightly.
Let stand at room temperature for 24 hours, then store at 4 ° C until measurement.

3. First reaction After removing the solution in the well of each synthetic MDM2 partial peptide antigen-immobilized plate by suction, the serum of a patient considered to have a high MDM2 autoantibody titer was reduced to 0.02% Antifoam.
Dilute 9-fold with 0.05 M PBS (pH 7.4) containing A, add 100 μl to each well, and allow to react at room temperature for 2 hours. 4. Second reaction After the first reaction, the solution in the well is removed by suction, and each well is washed twice with 2 ml of physiological saline containing 0.05% Tween 20 using a microplate washer. Then each M
Rabbit polyclonal anti-MDM2 antibody (IgG) against each solid-phased partial peptide antigen prepared individually using the DM2 partial peptide as an immunogen (1.
Is adjusted to 20 ng / ml in 0.05 M PBS (pH 7.4) containing 1% normal rabbit serum, and 100 μl is added to each well, followed by reaction at room temperature for 2 hours.

5. Third reaction After the second reaction, the solution in the well is removed by suction, and each well is washed twice with 2 ml of physiological saline containing 0.05% Tween 20 using a microplate washer. Then, Pe
roxidase-Monoclonal Rat a
anti-Rabbit IgG (ZYMED Lab.
Inc. PB) containing 1% rat serum
Dilute 1000-fold (450 ng / ml) with S (pH 7.4), add 100 μl to each well, and allow to react at room temperature for 45 minutes. 6. Fourth reaction After the third reaction, the solution in the well is removed by suction, and each well is washed twice with 2 ml of physiological saline containing 0.05% Tween 20 using a microplate washer. Next, a 0.1 M citrate phosphate buffer solution (2 mg / ml of 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt, manufactured by Nacalai Tesque) was used as a coloring agent ABTS (Nacalai Tesque). (pH 4.0), a 20% aqueous hydrogen peroxide solution as a substrate was added thereto to a concentration of 0.04%, and 100 μl of this solution was added to each well, followed by reaction at room temperature for 30 minutes.

7. Termination of reaction After completion of the fourth reaction, 0.1% sodium azide was added.
The reaction is stopped by adding 100 μl of 1 M citrate phosphate buffer (pH 4.0) per well. The color of each well is measured for absorbance at a wavelength of 420 nm. 8. Selection of MDM2 partial peptide antigen From among the MDM2 partial peptide antigens, MDM2 / N with the lowest absorbance of the test serum was selected as the antigen, and used for the measurement of MDM2 autoantibodies in the sample shown in Example 2. I made it.

Example 2 Measurement of MDM2 Autoantibody by Inhibition Method Rabbit polyclonal anti-MDM2 antibody against the MDM2 / N antigen prepared in Example 1 was prepared, and MD method was performed by the inhibition method.
The measurement of M2 autoantibody was performed.

1. Preparation of rabbit polyclonal anti-MDM2 antibody (IgG) MDM2 / N prepared in Example 1 was replaced with Freund's Complete Adjuvant (Freund's Complete Adjuvant).
After the mixture was mixed with the same amount and sufficiently emulsified, it was administered to rabbits for immunization. Immunization was performed every two weeks, and antiserum obtained by collecting blood four months later was subjected to 40% ammonium sulfate fractionation, followed by dialysis and concentration, and then rabbit polyclonal anti-MDM2 antibody (IgG) by protein A affinity chromatography.
I got

2. Preparation of MDM2 antigen-immobilized microtiter plate The microtiter plate (AquaBind, AquaBind, MDM2 / N) prepared in Example 1 was adjusted to a concentration of 5 μg / 150 μl / well with 0.1 M carbonate buffer (pH 9.0).
(M & E, Denmark). After reacting at room temperature for 3 hours, the antigen solution in each well was removed by suction, and a microplate washer (Minilab Washer, manufactured by Lifetec) was used.
Wash each well twice with 1 ml of 0.1 M carbonate buffer (pH 9.0). Then, 0.05M PBS containing 15% polyethylene glycol (MW4000), 10 mM diethanolamine and 0.1% sodium azide.
(PH 7.4) was added at a rate of 250 μl / well, and the seal was firmly kept, allowed to stand at room temperature for 24 hours, and then stored at 4 ° C. until measurement.

3. Preparation of Anti-MDM2 Antibody Standard Solution An anti-MDM2 antibody standard solution was prepared from the rabbit polyclonal anti-MDM2 antibody (IgG) prepared above using MDM2 / N as an immunogen as follows. That is, a rabbit polyclonal anti-MDM2 antibody (IgG) was added to 0.05 ng / PBS (pH 7.4) containing 1% normal rabbit serum at a concentration of 20 ng / g.
1.25 ng with the same buffer.
/ Ml, and dilute it to 5 concentrations of anti-MDM2.
An antibody standard solution was used. A 0 ng / ml anti-MDM2 antibody standard solution was prepared from 0.05 M PBS (pH: 1%) containing 1% normal rabbit serum.
7.4) was used as is.

4. Measurement of MDM2 autoantibody in blood 1) First reaction 15% polyethylene glycol (MW4000), 10 mM diethanolamine and 0.1% sodium azide in each well of the MDM2 / N antigen-immobilized microtiter plate prepared in 2 above 0.05M PB containing
S (pH 7.4) is removed by suction. Without the test serum, the wells containing the anti-MDM2 antibody standard solution in the second reaction were 0.05M P containing 0.1% sodium azide.
BS (pH 7.4) was added in 100 μl portions. To the other wells, 100 μl of test serum diluted 9-fold with 0.05M PBS (pH 7.4) containing 0.02% Antifoam A is dispensed per well. Incubate at room temperature for 2 hours. 2) Second reaction After the completion of the first reaction, the solution in the wells is removed by suction, and each well is washed twice with 2 ml of physiological saline containing 0.05% Tween 20 using a microplate washer. Then
The 20 ng / ml anti-MDM2 antibody standard solution prepared in 3 above was added to each well in which the test serum was reacted in the first reaction.
Add in μl. In addition, the above-mentioned 3. Anti-M at each concentration prepared in
Dispense 100 μl of DM2 antibody standard solution per well. Incubate at room temperature for 2 hours.

3) Third reaction After completion of the second reaction, the solution in the well is removed by suction, and each well is washed twice with 2 ml of a physiological saline solution containing 0.05% Tween 20 using a microplate washer. Then
Peroxidase-Monoclonal Rat
anti-Rabbit IgG (ZYMED La
b. Inc. 0.05% containing 1% rat serum
1000 times (450 ng / m2) with PBS (pH 7.4)
1), add 100 μl to each well, and react at room temperature for 45 minutes. 4) Fourth reaction After completion of the third reaction, the solution in the well is removed by suction, and each well is washed twice with 2 ml of a physiological saline solution containing 0.05% Tween 20 using a microplate washer. Then
Coloring agent ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt,
Nacalai Tesque) to 3 mg / ml.
M was dissolved in citrate phosphate buffer (pH 4.0), 20% hydrogen peroxide solution was added as a substrate so that the concentration became 0.04%, and 100 μl of this solution was added to each well.
Incubate at room temperature for 30 minutes.

5) Termination of reaction After completion of the fourth reaction, 0.1% sodium azide containing 0.1% sodium azide was added.
The reaction is stopped by adding 100 μl of 1 M citrate phosphate buffer (pH 4.0) per well. 6) Measurement of absorbance The color of each well is measured for absorbance at a wavelength of 420 nm. 7) Determination of MDM2 autoantibody concentration A standard curve was drawn from the absorbance of the anti-MDM2 antibody standard solution, the absorbance of each well was applied to the standard curve, and the autoantibody in the test serum was bound.
The concentration at which the binding of ng / ml was inhibited was determined, and this was defined as the concentration of MDM2 autoantibody in blood.

8) Results of measurement of healthy subjects and various cancer patients using the above-described inhibition method From the measurement results of blood MDM2 autoantibodies of healthy subjects,
The mean value of 2 cases ± 2 SD (twice the standard deviation) is 8.2 ± 1
At 1.9 ng / ml, all 20 cases were below 20 ng / ml. On the other hand, the mean ± 2SD for women is 2 in this example.
At 7.5 ± 18.6 ng / ml, 19 out of 20 cases were 15n
g / ml or more showed significantly higher antibody measurement values compared to males. Since there was a significant difference in the distribution of antibody measurement values between healthy men and women, the cutoff value for both men and women was provisionally set to 15 ng / ml, and the positive rate was calculated as positive for men and above the cutoff value. For women, the positive rate was calculated as the positive value below the cutoff value. That is, anti-MDM2 autoantibody measured values of each test serum were tabulated separately for males and females.

Table 1 Measurement results of MDM2 autoantibodies in various male cancer patients 疾患 Disease Examples Number Mean ± 2SD Positive rate Significance test ng / ml% (vs. healthy person) ─ Healthy person 20 8.2 ± 11.9 10 − Colorectal cancer 11 24.9 ± 17.4 100 P <0.001 Gastric cancer 19 20.6 ± 22.8 68 P <0.001 Pancreatic cancer 4 15.7 ± 7.9 50 P <0.01 Hepatocellular carcinoma 4 29.0 ± 23.8 75 P <0.05 Lung cancer 11 28.5 ± 22.0 82 P <0.001───────── ───────────────────────

Table 2 Measurement results of MDM2 autoantibodies in various female cancer patients ──────────────────────────────── Disease Examples Number Mean ± 2SD Positive rate Significance test ng / ml% (vs. healthy person) ─ Healthy person 20 27.5 ± 18.6 5 − Breast cancer 27 13.2 ± 22.5 52 P <0.001 Colorectal cancer 5 22.5 ± 20.0 40 NS Gastric cancer 3 21.2 ± 35.5 33 NS pancreatic cancer 1 20.30 NS hepatocellular carcinoma 2 18.8 ± 5.60 NS ovarian cancer 2 19.7 ± 1.80 NS NS lung cancer 4 26.9 ± 26.0 25 NS ────── Ｎ NS: No significant difference

Tables 1 and 2 show the mean ± 2SD of the measured values of MDM2 autoantibody in blood for each gender. Male cancer patients tended to have a statistically significantly higher mean ± 2SD in all of colon cancer, stomach cancer, pancreatic cancer, hepatocellular carcinoma, and lung cancer than healthy subjects. On the other hand, in this example, there were no cases in which the mean value ± 2SD was significantly higher in female cancer patients than in healthy subjects, and conversely, the average value ± 2SD was significantly lower in breast cancer patients than in healthy female subjects. The tendency was significantly lower (P <0.001).
The positive rate of MDM2 autoantibody in the blood of men was 100% in 11 cases of colorectal cancer, 68% in 19 cases of gastric cancer, 50% in 4 cases of pancreatic cancer, 75% in 4 cases of hepatocellular carcinoma, and 82% in 11 cases of lung cancer. there were. The positive rate for cancer diseases was sufficiently higher than the positive rate for healthy men of 10%.

Embodiment 3 FIG. Measurement of MDM2 Autoantibody by Competition Method (1) MDM2 autoantibody was measured by a competition method using biotinylated MDM2 / N in which the N-terminal was biotinylated.

1. Preparation of avidin-immobilized microplate Avidin (manufactured by Nacalai Tesque) was adjusted to a concentration of 30 μg / ml in a 0.1 M sodium bicarbonate buffer solution (pH 9.
6), and 100 μl thereof was transferred to a microplate (1).
x8 strip well plate, CORNING Co
(manufactured by Star Co., Ltd.) and left at 4 ° C. overnight. Thereafter, the avidin solution was discarded, and 0.05 M PBS (pH 7.2) containing 1% bovine serum albumin was added to 300 ml of the avidin solution.
The solution was added at a rate of μl / well, sealed, and allowed to stand at 4 ° C. overnight, and then stored at 4 ° C. until measurement.

2. Biotinylated M with N-terminal biotinylated
Preparation of DM2 / N The N-terminal amino acid (asparagine) of the MDM2 / N was biotinylated with N-succiimidyl D-biotin according to a conventional method to prepare a biotinylated MDM2 / N peptide. Purification was performed by reversed-phase HPLC, and the purity was 97%.
% Biotinylated MDM2 / N.

3. Measurement of MDM2 autoantibody in blood 1) First reaction 2% dextran 70000 (manufactured by Tokyo Chemical Industry), 3% bovine serum albumin, 0.9% Triton X
9 with 0.05M PBS (pH 7.4) containing -100
Add 100 μl of the test sample dilution diluted in double to the test tube.
The rabbit polyclonal anti-MDM2 antibody (IgG) prepared in Example 2 was adjusted to 20 ng / ml with 2% dextran 70000, 3% bovine serum albumin and 0.9%
0.05 M PBS containing Triton X-100 (p
H7.4). 100 μl of the obtained competitive antibody solution
Is added to the test tube containing the test sample solution. Furthermore, biotinylated MDM2 / N 19.5 with N-terminal biotinylated
ng / ml containing 0.05M PBS (pH 7.4) 10
Add 0 μl. Immediately mix well and leave at room temperature for 2 hours.

2) Second reaction The avidin-immobilized microplate prepared above was returned to room temperature, the solution in the well was removed by suction, and 0.05% Tw was added.
Phosphate physiological saline (pH 7.4) containing een20 3m
Wash each well three times with l. The reaction solution after the first reaction is dispensed 100 μl per well. Set in a microplate incubator (TOMY) and set to 30
Incubate at 90 ° C for 90 minutes. 3) Third reaction After the completion of the second reaction, the solution in the well is removed by suction, and each well is washed three times with 3 ml of PBS (pH 7.4) containing 0.05% Tween 20 using a microplate washer. Next, Peroxidase-Monoclon
al Rat anti-Rabbit IgG (ZY
MED Lab. Inc. Made from 1% rat serum and 1% bovine serum albumin in 0.05M PBS (pH
The solution is diluted 500 times (7.4 μg / ml) in 7.4), added to each well in an amount of 100 μl, set in a microplate incubator, and reacted at 30 ° C. for 45 minutes.

4) Fourth reaction After completion of the third reaction, the solution in the well was removed by suction, and each well was washed three times with 3 ml of PBS (pH 7.4) containing 0.05% Tween 20 using a microplate washer. I do. Next, the coloring agent ABTS was dissolved in a 0.1 M citrate phosphate buffer (pH 4.0) to a concentration of 3 mg / ml, and a 20% aqueous hydrogen peroxide solution as a substrate was added thereto in an amount of 0.04%.
100 μl of this solution is added to each well and allowed to react at room temperature for 10 minutes. 5) Stopping the reaction After the fourth reaction, add 100 μl of 0.1% aqueous sodium azide solution to each well to stop the reaction. I do. 6) Measurement of absorbance The absorbance of each well is measured at a wavelength of 415 nm.

7) Method for Determining MDM2 Autoantibody Concentration in Test Sample In this method, the higher the MDM2 autoantibody concentration in the sample, the lower the absorbance. Therefore, based on the absorbance measured when a sample containing no MDM2 autoantibody was measured, the percentage of inhibition of color development was defined as the measured value of the autoantibody concentration. That is, a test sample diluent containing no test sample serum and 20 ng
/ Ml rabbit polyclonal anti-MDM2 antibody (IgG)
A. The absorbance obtained by using a competitive antibody solution containing the test sample A was measured using a test sample diluent containing no test sample serum and a competitive antibody solution containing no rabbit polyclonal anti-MDM2 antibody (IgG). When the obtained absorbance is B, and the absorbance obtained by measuring the absorbance obtained by using a test sample diluent containing the test sample serum and a competitive antibody solution containing 20 ng / ml rabbit polyclonal anti-MDM2 antibody (IgG) is defined as C The value defined by the following formula was used as a measured value indicating the concentration of the autoantibody. Measurement value (%) = {(AC) / (AB)} × 100

8) Results of Measurement FIG. 1 shows the results of measurement by the competition method. In this example, there were no significant differences in the measured values between the two healthy subjects in eight males and eight females. The MDM2 autoantibody concentration in the blood of a healthy person was 5% or less in inhibition rate, and the cutoff value was 5.1% or more. Cancer patients showed a high inhibition rate regardless of gender, with colon cancer 37.5%, gastric cancer 87.5%, esophageal cancer 100%, lung cancer 100%, pancreatic cancer 100%, liver cancer 100%, gallbladder cancer 100
%, Prostate cancer 100%, and breast cancer 50%, showing good results.

Embodiment 4 FIG. Measurement of MDM2 Autoantibody by Competition Method (2) In Example 3, a measurement method by a competition method in a liquid layer was examined. This example is a measurement method based on a competitive reaction between a biotinylated rabbit polyclonal anti-MDM2 antibody against immobilized MDM2 / N antigen and an MDM2 autoantibody present in serum, and the standard antibody is a rabbit polyclonal anti-MDM2 antibody. And using a test serum sample different from that in Example 3.

1) Biotinylated rabbit polyclonal anti-MD
Preparation of M2 Antibody The rabbit polyclonal anti-MDM2 antibody prepared in Example 2 was added to a 50 μM PBS buffer (pH 7.8) at 50 μL.
g / 250 μl, and a reagent for introducing a biotin group into an amino group, EZ-Link ^™ S
ulfo-NHS-LC-Biotin (PIERCE
Is prepared with distilled water to a concentration of 2 mg / 50 μl, and both are reacted in a glass tube at room temperature of 25 ° C. for 3 hours under light shielding. After the reaction, D-Salt ^™ Desa
The reaction solution is passed through an lting Column (manufactured by PIERCE) to terminate the reaction. Then, excess EZ-Li
nk ^™ Sulfo-NHS-LC-Biotin reagent,
Rabbit polyclonal anti-MDM2 not biotinylated
The antibody and the biotinylated rabbit polyclonal anti-MDM2 antibody are separated and eluted (0.5 ml / fraction) with a 0.05 M PBS buffer (pH 7.4). The fractions of the biotinylated rabbit polyclonal anti-MDM2 antibody were selected from the respective fractions by the following method.

Using the MDM2 / N antigen-immobilized microtiter plate prepared in Example 2, each column chromatographic fraction was treated with 1% fetal calf serum (FCS) in the same buffer as used for elution of the fraction. ) Was diluted 500-fold with a buffer containing
The reaction is carried out at 30 ° C. for 60 minutes. After completion of the reaction, the reaction solution in the well was removed by suction, and 0.02% Tween was removed.
Wash three times with saline containing en20. Then 1
_{% FCS, 1mM MgCl 2, 0.9} % NaCl,
50 mM Tris containing 0.1% sodium azide
Alkaline phosphatase-labeled streptavidin (manufactured by ZYMED Lab. Inc.) adjusted to a concentration of 3.0 μg / ml with an HCl buffer (pH 8.0) is dispensed in 100 μl portions into each well, and reacted at 30 ° C. for 30 minutes. .
After completion of the reaction, the reaction solution in the well is removed by suction, and each well is washed three times with a physiological saline solution containing 0.02% Tween20. Then 0.1M with 1 mM MgCl ₂
Disodium paranitrophenylphosphate (manufactured by Nacalai Tesque) adjusted to a concentration of 4.0 mg / ml with a carbonate buffer (pH 9.6) was dispensed into each well in an amount of 100 μl.
Incubate at 0 ° C. for 30 minutes. After the reaction is complete, add 1 well to each well.
The reaction is stopped by adding 100 μl each of N sodium hydroxide, and the absorbance of each well is measured at a wavelength of 405 nm. A fraction having a high absorbance is selected and used for measurement as a biotinylated rabbit polyclonal anti-MDM2 antibody.

2) First reaction In order to prepare a test serum or a standard curve used for quantification, a rabbit polyclonal anti-MDM2 antibody having seven concentration series from 32 μg / ml to 0.5 μg / ml was prepared by
0.05MP containing 0.1% FCS and 0.1% normal rabbit serum
Each was diluted 9-fold with a biotinylated rabbit polyclonal anti-MDM2 antibody solution adjusted to a concentration of 133 ng / ml with a BS buffer (pH 7.4), and 100 μl of each was diluted in Example 2.
Dispense into each well of the MDM2 / N antigen-immobilized microtiter plate prepared in the above and react at 30 ° C. for 2 hours. 3) Second reaction After completion of the first reaction, the reaction solution in the wells was removed by suction, and each well was washed with a physiological saline solution containing 0.02% Tween20.
Wash twice. Then, 1% FCS, 1 mM MgC
50 mM Tris · HCl buffer (pH 8.0) containing l ₂ , 0.9% NaCl and 0.1% sodium azide.
0) to a concentration of 3.0 μg / ml in alkaline phosphatase-labeled streptavidin (ZYMED L).
ab. Inc. Was added to each well in an amount of 100 μl.
Incubate at 30 ° C. for 30 minutes.

4) Third Reaction After the completion of the second reaction, the reaction solution in the wells is removed by suction, and each well is washed with a physiological saline solution containing 0.02% Tween 20 for 3 hours.
Wash twice. Then 0.1 mM MgCl ₂ containing 0.1 mM
100 μl of disodium paranitrophenylphosphate (manufactured by Nacalai Tesque) adjusted to a concentration of 4.0 mg / ml with M carbonate buffer (pH 9.6) is dispensed into each well, and reacted at 30 ° C. for 30 minutes. . 5) Stopping the reaction After completion of the third reaction, add 1N sodium hydroxide to each well.
Stop the reaction by adding 00 μl each.

6) Measurement of Absorbance The absorbance of each well is measured at a wavelength of 405 nm. 7) Determination of MDM2 Autoantibody Concentration The absorbance of a rabbit polyclonal anti-MDM2 antibody standard solution having seven concentration series measured to create a standard curve is plotted on the vertical axis, and the concentration is plotted on the horizontal axis. I drew.
One example is shown in FIG. Using the standard curve, the measured absorbance of each test serum was applied, and the blood MD in the test serum was determined.
The M2 autoantibody concentration was determined.

8) Results of Measurement FIG. 3 shows the results of measurement of MDM2 autoantibody in blood. Blood MDM2 in a total of 49 cases of 24 healthy men and 25 women
Autoantibodies showed 5 μg / ml or less. When this value is used as a cutoff value, the positive rates of blood MDM2 autoantibodies in cancer patients are 63.9% (23/36) for breast cancer, 33.3% (17/51) for gastric cancer, and 65 for lung cancer. .2% (30 /
46). Blood MDM2 in Breast and Gastric Cancer by Stage
The positive rate of autoantibodies is shown in FIGS. Stage 0 1 for breast cancer
00% (1/1), Stage I 80% (8/10), Stage II 58.
The positive rates were 8% (10/17), stage III 50% (4/8), and in gastric cancer, stage IA was 34.3% (12/35), stage IB was 44.4% (4/9), II Phase 25% (1/4), Phase III 0
% (0/3). It was found that the positive rates at stage 0 and stage I for breast cancer and stage IA and stage IB for gastric cancer tended to be higher than those at stage II or stage III.
It was suggested that M2 autoantibody measurement was useful for early diagnosis of breast cancer and gastric cancer.

FIG. 6 shows the measurement results of MDM2 autoantibody in blood by lung cancer tissue type. 75% (9/1) in squamous cell carcinoma
2), adenocarcinoma 60% (18/30), small cell carcinoma 100%
(2/2), the positive rate was 0% (0/1) for large cell carcinoma. In addition, the blood M according to the degree of differentiation of lung squamous cell carcinoma and lung adenocarcinoma
FIG. 7 shows the measurement results of the DM2 autoantibody. The positive rate of MDM2 autoantibodies in blood by lung differentiation squamous cell carcinoma was 100% (1/1) in the well-differentiated type and 100% (7 /
7), 25% (1/4) poorly differentiated, 83% (5/6) highly differentiated, 47% (7/1)
5) Positive rate of poorly differentiated 0% (0/1), which is more differentiated in lung squamous cell carcinoma and lung adenocarcinoma than poorly differentiated
The positivity of MDM2 autoantibodies in blood tended to be higher. From the results of immunohistological staining using rabbit polyclonal anti-MDM2 antibody in the same case of lung squamous cell carcinoma and lung adenocarcinoma, well-differentiated type was more The expression of the MDM2 antigen was found to be high. FIG. 8 shows the measurement results of MDM2 autoantibodies in blood according to the degree of differentiation of gastric cancer. Blood MDM2 according to differentiation level in gastric cancer
The positive rate of autoantibodies was 36.7% for the differentiated type (11/3).
0) and 28.6% (6/21) of the undifferentiated type. In gastric cancer, the positive rate of the blood MDM2 autoantibody was slightly higher in the differentiated type than in the undifferentiated type. Also in this example, as in Example 3, cancer patients showed higher values than healthy individuals, indicating that cancer can be diagnosed by the present invention.

[0066]

According to the present invention, autoantibodies to MDM2 in blood can be quantitatively measured simply and with high sensitivity, and the value can be used as an index to detect cancer more specifically and early. Diagnosis of cancer becomes easier. That is, autoantibodies to MDM2 are also present in the blood of healthy individuals, but generally increase significantly in the blood of cancer patients, and can be used for cancer diagnosis. MDM2 of healthy people and cancer patients
Since the concentration distribution of the autoantibodies is different, a certain value (cutoff value) is set and compared with the value, thereby enabling cancer diagnosis.

[0067]

[Sequence List] SEQUENCE LISTING <110> NIPPON KAYAKU KABUSHIKI KAISHA <120> Detection of cancer by measurement of autoantibody to MDM2, and the reagent reagent <130> NKM1839 <150> JP 2000-93569 <151> 2000-3-30 <160> 1 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> PRT <213> Homo sapiens <400> 1 Asn Thr Asn Met Ser Val Pro Thr Asp Gly Ala Val Thr Thr Ser Gln 1 5 10 15 Ile Pro Ala Ser 20

[0068]

[Brief description of the drawings]

FIG. 1 shows results of measurement of blood MDM2 autoantibodies by the competition method of Example 3.

FIG. 2 Rabbit polyclonal anti-MDM2 antibody standard curve

FIG. 3 shows results of blood MDM2 autoantibody measurement by the competition method of Example 4.

FIG. 4. Measurement results of MDM2 autoantibodies in blood by stage of breast cancer

FIG. 5: Measurement results of MDM2 autoantibodies in blood by gastric cancer stage

FIG. 6: Measurement results of MDM2 autoantibodies in blood by lung cancer tissue type

FIG. 7: Blood MD by differentiation degree of lung squamous cell carcinoma and lung adenocarcinoma
Measurement results of M2 autoantibody

FIG. 8: Measurement results of blood MDM2 autoantibodies by gastric cancer differentiation level

Claims (20)

[Claims] 1. A method for testing a cancer, comprising measuring an autoantibody against MDM2 present in a sample. 2. The test method according to claim 1, wherein the cancer is early cancer. 3. The method according to claim 1, wherein the cancer is epithelial cancer. 4. The test method according to claim 3, wherein the epithelial cancer is lung squamous cell carcinoma, lung adenocarcinoma or small cell lung cancer. 5. The test method according to claim 1, wherein the measurement of the autoantibody is performed by an immunological measurement method. 6. The test method according to claim 5, wherein the immunological measurement method is a competition method, an inhibition method or a sandwich method. 7. The method according to claim 7, wherein the antigen used in the immunological assay is MDM.
The test method according to claim 5 or 6, wherein the test peptide is a partial peptide of (2). 8. The test method according to claim 5, wherein an anti-MDM2 antibody or a label thereof is used. 9. The test method according to claim 8, wherein the anti-MDM2 antibody is an antibody obtained by using a partial peptide of MDM2 as an antigen or a modified product thereof. 10. The method according to claim 10, wherein the partial peptide of MDM2 is N
The test method according to claim 7, which is a partial peptide of a terminal region or a modified product thereof. 11. The test method according to claim 9, wherein the partial peptide of MDM2 is immobilized on a solid phase. 12. A peptide represented by the following (a) or (b): (a) a peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and having a function of binding to an MDM2 autoantibody (b) A) a peptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing in which some amino acids have been deleted, substituted or added, and having a function of binding to an MDM2 autoantibody; 13. A cancer test reagent comprising an anti-MDM2 antibody or a labeled product thereof. 14. A reagent for cancer test comprising MDM2, its partial peptide or a labeled product thereof. 15. The method according to claim 15, wherein the partial peptide of MDM2 is N
The cancer test reagent according to claim 14, which is a partial peptide of a terminal region or a modified product thereof. 16. The reagent according to claim 14, wherein the partial peptide of MDM2 is immobilized on a solid phase. 17. The partial peptide of MDM2 has the following (a)
Or a peptide represented by (b): (a) a peptide having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and having a function of binding to an MDM2 autoantibody; (b) a peptide represented by SEQ ID NO: 1 in the sequence listing The peptide according to any one of claims 14 to 16, which is a peptide having an amino acid sequence in which some amino acids are deleted, substituted, or added in the amino acid sequence, and which has a function of binding to an MDM2 autoantibody. Cancer testing reagents 18. The reagent according to claim 13, wherein the cancer is epithelial cancer. 19. A cancer test kit comprising the cancer test reagent according to any one of claims 13 to 18. 20. The method according to claim 19, further comprising a standard solution for preparing a standard curve.
Cancer testing kit described in