JP2003315341A - Diagnostic method of toxemia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simplify a toxemia diagnosis by providing a marker protein recognized in the human organism in a toxemia pathology. <P>SOLUTION: This diagnostic method of toxemia and a blood viscosity abnormality includes reaction among a copper ion immobilizing chip, an anion exchange chip and/or a cation exchange chip and human body fluid, analysis by mass spectrometry of a protein interacting with the chip, and comparison of the analytical result between a patient and a healthy person. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a marker for diagnosing blood and a method for diagnosing blood. In addition, although "in blood" may be written in the following Chinese characters, it is abbreviated as "in blood" in this specification.

[0002]

[Chemical 1]

[0003]

2. Description of the Related Art "In blood" is one of the most important pathological conditions in oriental medicine, and "the blood that should be circulated smoothly in the blood vessel is caught for some reason and cannot be circulated smoothly. Pathology ”(Hozo Shibasaki, Clinical Chinese Medicine, 16
(7), 1969) and is recognized as a syndrome that includes blood circulation disorders. Blood is thought to be a condition deeply involved in various diseases,
By treating blood symptoms, it is expected to reduce the symptoms and prevent the onset of the disease. Studies to date have shown an increase in intravascular red blood cell aggregation (IEA) in blood,
It has been reported that the maximum diameter of red blood cell aggregates (DEA) in blood vessels is increased, the viscosity of blood is increased, and the amount of fibrinogen, which influences the ability to collect red blood cells, is increased (Koji Terazawa, The Journal of Oriental Medicine, 48 (4), 1998 ).

The increase in blood viscosity observed in blood is known as a risk factor for developing serious diseases such as cerebrovascular disorders, which are thought to be deeply related to lifestyle-related diseases, and so-called economy class syndrome. Normalizing the viscosity is also considered to be important in preventive medicine.

Currently, the blood pathological condition is the blood blood diagnostic criteria by the research group of the Science and Technology Agency (Koji Terasawa et al., The Journal of Japanese Oriental Medicine, 34
(1), 1983) evaluated by the score calculated according to
The determination of the therapeutic effect is also made based on the change in the score, but no diagnostic marker has been found or reported. As a result, there are variations in blood diagnosis among doctors,
Some skill is required to make a diagnosis.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a marker protein found in human body for blood pathological conditions,
The task to be solved is to simplify blood diagnosis.
Furthermore, the present invention provides a marker protein whose expression varies before and after blood therapy, and aims at facilitating follow-up observation.

[0007]

[Means for Solving the Problems] The inventors of the present invention have made extensive studies in order to solve the above-mentioned problems. First, a blood score is calculated according to the blood diagnosis criteria by the research group of the Ministry of Science and Technology, and the blood is diagnosed. The proteins contained in the plasma of patients and the plasma of healthy subjects were comprehensively analyzed using a protein chip system. At the same time, changes in plasma proteins before and after the blood treatment were analyzed in the same manner. Furthermore, blood rheological measurement was performed at the same time as the blood score calculation to correlate. As a result, we succeeded in identifying a marker protein useful for blood diagnosis, and completed the present invention.

That is, according to the present invention, a copper ion-immobilized chip, an anion exchange chip and / or a cation exchange chip are reacted with a human body fluid, and the protein interacting with the chip is analyzed by mass spectrometry, Provided is a method for diagnosing abnormal blood or blood viscosity, which comprises comparing the analysis results of a patient and a healthy person.

According to a preferred embodiment of the present invention, (1) a copper ion-immobilized chip is reacted with human body fluid, and the protein interacting with the chip is analyzed by mass spectrometry to determine m / z
At 3865 (3861-3865), 3904, 3929, 4411, 4547 (4542-454
7), 8086, 9251 (9251-9277), 9910 (9902-9910), 14929
(14919-14929), 15087 (15079-15087), 15288 (1528
8-15294), 15827 (15824-15827), 16992, 17183, 182
93, 19835 (19815-19839), 20016, 28177 (28094-2819
4), 28900 (28856-28909), 30514, 33368 (33274-3339)
Detecting the presence or amount of any one or more proteins having 5), 60191, or 66606 (66507-66620), or (2)
The anion-exchange chip and human body fluid were reacted, and the proteins that interacted with the chip were analyzed by mass spectrometry.
At 3906 (3905-3906), 3918 (3917-3921), 4124 (4119
-4124), 28092 (28084-28178), 4062 (4059-4062),
5445 (5444-5445), 5601, 8414 (8404-8414), 8520 (8
508-8520), 8641 (8632-8650), 9136 (9131-9142), 929
9 (9288-9299), 13687 (13681-13707), 15065 (15039)
-15068), 15857 (15856-15881), or 16015 (16015-1)
Detecting the presence or amount of any one or more proteins having 6049), or (3) reacting the cation exchange chip with human body fluid and analyzing the protein interacting with the chip by mass spectrometry , M / z 4063, 4568 (4564-456
8), 9154 (9146-9154), 9310 (9302-9311), 13713 (1
3699-13713), 15065 (15054-15075), 15815, 15889 (1
5879-15894), 16033, 16086, 16221, 3901, 4278, 441
0 (4409-4410), 4619, 6156 (6155-6156), 6242 (624
2-6245), 7531 (7521-7533), 7737 (7725-7746), 78
98 (7898-7900), 7940 (7940-7942), 8111 (8107-811)
1), 8573 (8567-8573), 8782 (8776-8782), 8903 (8
902-8904), 9257 (9242-9263), 9466 (9465-9466),
10231 (10224-10231), 12819 (12801-12819), 14917, 1
5083 (15062-15084), 15279, 15814 (15814-15827),
Methods of diagnosing blood or blood viscosity abnormalities are provided that detect the presence or amount of any one or more proteins having 15885 or 16042. According to one aspect of the diagnostic method of the present invention, it can be performed for observing the therapeutic effect of blood or blood viscosity abnormality. According to another aspect of the diagnostic method of the present invention, it can be performed for diagnosing a disease characterized by abnormal blood or blood viscosity and / or observing a therapeutic effect. Preferably, diseases characterized by abnormal blood or blood viscosity are cerebrovascular disorders, coronary artery diseases, chronic hepatitis, rheumatoid arthritis, Behcet's disease, menstrual disorders / infertility, menopause, renal sclerosis, or peripheral vascular disorders. Is. In addition, in the present specification, m / z is a peak value detected by mass spectrometry, and when there is a deviation between samples even though the peaks are the same, a representative value is shown and the width in parentheses is shown. showed that.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below. The method for diagnosing abnormal blood or blood viscosity according to the present invention comprises reacting a copper ion-immobilized chip, an anion exchange chip and / or a cation exchange chip with a human body fluid, and measuring the protein interacting with the chip by mass spectrometry. It is characterized by analyzing and comparing the analysis results of a patient and a healthy person.

No marker protein has been reported so far which is useful for diagnosis of blood pathological conditions. The present inventors analyzed proteins of blood patients and healthy individuals using a copper ion-immobilized chip, an anion exchange chip and / or a cation exchange chip for analyzing protein interactions. It was the first discovery that the expression states of proteins differ between body fluids of patients and healthy people. The body fluid referred to in the present specification means any body fluid including blood, plasma, serum, saliva, sweat, urine and the like. Known diseases including blood or blood viscosity abnormality as risk factors include cerebrovascular disease, coronary artery disease such as angina pectoris / myocardial infarction, chronic hepatitis, rheumatoid arthritis, Behcet's disease, menstruation. Disorders / infertility, menopausal disorders, renal sclerosis, and peripheral vascular disorders such as chronic arteriosclerosis obliterans / thrombophlebitis. That is, it is important to detect abnormal blood or blood viscosity in diagnosing and treating the above diseases.

In the method for diagnosing abnormal blood or blood viscosity according to the present invention, a copper ion-immobilized chip, an anion exchange chip and / or a cation exchange chip are used. The copper ion-immobilized chip is a solid phase carrier having copper ions on its surface. The anion exchange chip is a solid phase carrier having a functional group having an anion exchange action on its surface. The cation exchange chip is a solid phase carrier having a functional group having a cation exchange action on its surface.

Specific examples of the above chip which can be used in the present invention include CIPHERGEN used in the following examples.
A protein chip system manufactured by the company can be used. The analysis method using this protein chip system is shown below. (1) Addition of sample 1 to several hundred μL of sample (plasma) is added to a spot having a diameter of about 2 mm. (2) Washing After incubation, the surface is washed with water or a buffer solution to remove substances having no affinity on the chip surface. As a result, it is possible to remove the protein that is not bound to the chip surface, as well as salts and surfactants that interfere with the measurement of the target protein. (3) Addition of energy absorbing molecule (EAM) Energy absorbing molecule (EAM) necessary for measuring the target protein
AM) and dry.

(4) Measurement The sample on the protein chip is measured by a time-of-flight mass spectrometer. By irradiating the protein chip with a UV pulsed laser, the energy-ionized protein is accelerated at a constant voltage and flies toward an ion detector opposite the vacuum tube. At this time, the lighter the molecule, the faster it takes to reach the ion detector tube.
The heavier the molecule, the slower it is, so the mass number of a substance can be determined by measuring the time of flight. (5) Protein expression analysis According to the present method, it is possible to simultaneously measure a large number of proteins captured on a protein chip, and thus it is possible to analyze the expression of proteins in a sample. It is also possible to directly compare protein expression analyzes between different samples (eg healthy people and patients).

The diagnosis of blood or blood viscosity abnormality using these three types of chips will be described in detail below.

(1) Utilization of copper ion-immobilized chip According to the first aspect of the present invention, a copper ion-immobilized chip is reacted with human body fluid, and the protein interacting with the chip is analyzed by mass spectrometry. Then, the presence or amount of the following proteins is detected.

That is, 8086 (peak 6), 9251 (peak 7), 14929 (peak 9), 15087 (peak 1) at m / z found in healthy people.
0), 15288 (peak 11), 15827 (peak 12), 28177 (peak
18), 28900 (Peak 19), 30514 (Peak 20), 33368 (Peak 21), 60191 (Peak 22) or 66606 (Peak 23) If the peak has disappeared or decreased, blood or its The possibility can be predicted.

Further, at m / z, 3865 (peak 1), 3904 (peak)
2), 3929 (peak 3), 4411 (peak 4), 4547 (peak 5), 9
910 (peak 8), 16992 (peak 13), 17183 (peak 14), 18
293 (peak 15), 19835 (peak 16) or 20016 (peak 17)
When the peak having the number appears or increases more than that in a healthy person, blood or its possibility can be predicted.

Further, for m / z 3904 (peak 2), 3929 (peak 3), 4411 (peak 4), 19835 (peak 16), or 28900 (peak 19), the therapeutic effect by drug administration or the like is observed. It is also useful as a marker for

(2) Utilization of anion exchange chip According to the second aspect of the present invention, an anion exchange chip is reacted with human body fluid, and the protein interacting with the chip is analyzed by mass spectrometry, The presence or amount of the following proteins is detected.

Washing buffer is 50 mmol / L Sodium aceta
te (pH4), if the peak having 3906 (peak 25), 3918 (peak 26) or 28092 (peak 28) at m / z found in healthy people disappears or decreases, blood Or the possibility can be predicted.

The washing buffer was replaced with 50 mmol / L Tris-HCl (pH
8), 3906 (peak 2
9), 4062 (peak 30), 5445 (peak 31), 5601 (peak 3)
If the peak with 2), 8520 (peak 34) or 8641 (peak 35) disappears or diminishes, blood or its potential can be predicted. Also, at m / z 8414 (peak 33), 9136 (peak 36), 9299 (peak 37), 13687 (peak 38), 15065 (peak 39), 15857 (peak 40) or 160.
If the peak with 15 (peak 41) appears or is increased compared to a healthy person, blood or its potential can be predicted.

Furthermore, 4124 (peak 27), 28092 (peak 2)
The peak having 8), 8520 (peak 34) or 8641 (peak 35) is also useful as a marker for observing the therapeutic effect of drug administration or the like.

(3) Utilization of Cation Exchange Chip According to the third aspect of the present invention, a cation exchange chip and a human body fluid are reacted, and the protein interacting with the chip is analyzed by mass spectrometry, The presence or amount of the following proteins is detected.

Wash buffer with 50 mmol / L Sodium aceta
te (pH4) is 4063 at m / z observed in healthy people.
If the peak with (peak 42) disappears or diminishes, blood or its potential can be predicted. Also, at m / z 4568 (peak 43), 9154 (peak 44), 931
0 (peak 45), 13713 (peak 46), 15065 (peak 47), 158
15 (peak 48), 15889 (peak 49), 16033 (peak 50), 16
When a peak having 086 (peak 51) or 16221 (peak 52) appears or increases more than that in a healthy person, blood or its possibility can be predicted.

Washing buffer was added to 50 mmol / L Ammonium ace
When tate (pH 6) is used, it is 4278 at m / z that can be observed in healthy people.
(Peak 54), 4619 (Peak 56), 6156 (Peak 57), 6242
(Peak 58), 7737 (Peak 60), 8111 (Peak 63), 8573
(Peak 64), 8782 (Peak 65), 8903 (Peak 66), 9257
(Peak 67), 9466 (peak 68), 10231 (peak 69) or 149
If the peak with 17 (peak 71) disappears or diminishes, then blood or its potential can be predicted. Also, at m / z 3901 (peak 53), 4410 (peak 55), 753
1 (peak 59), 7988 (peak 61), 7940 (peak 62), 12819
(Peak 70), 15083 (Peak 72), 15279 (Peak 73), 1581
If a peak with 4 (peak 74), 15885 (peak 75) or 16042 (peak 76) appears or is increased over healthy individuals, blood or its potential can be predicted.

15889 (peak 49), 16086 (peak 5)
1), 16221 (peak 52), 3901 (peak 53), 4410 (peak 5)
5), 7988 (peak 61), 7940 (peak 62), 8782 (peak 6)
The peak having 5), 15814 (peak 74) or 16042 (peak 76) is also useful as a marker for observing the therapeutic effect of drug administration or the like.

In the present invention, by using a copper ion-immobilized chip, an anion exchange chip and / or a cation exchange chip, a marker protein useful for diagnosing blood or blood viscosity abnormality (hereinafter, the marker protein of the present invention is used. (Also referred to as)) was successfully identified for the first time. These marker proteins can be further purified and identified. The purification and identification methods will be described below.

(1) Purification Protein purification can be carried out by combining several types of protein chips with spin columns, electrophoresis and the like. (A) Fractionation by molecular weight: eg 3K, 30K, 70
Using a spin column of K or the like, the protein is roughly purified by utilizing the difference in molecular weight. (B) Purification with an ion exchange column: Purification is performed using a spin column packed with an ion exchange resin. After loading the sample, the pH of the elution buffer is changed to purify the protein by utilizing the difference in pI value of the protein.

(C) Purification using protein chip:
The sample purified using the spin column is further purified using a copper ion-immobilized chip or an ion exchange chip. At the time of cleaning, the pH of the ion exchange chip is changed and examined. (D) Electrophoresis: If necessary, further purification by electrophoresis is performed.

(2) Identification The protein purified in (1) above is digested with a proteolytic enzyme, and the digested fragment is captured on a normal phase protein chip to measure the molecular weight (peptide mapping).
Further, the protein is identified by inputting the measured molecular weight of the fragment into the software for protein identification and performing database analysis.

(A) Peptide mapping: The purified protein is decomposed on a protein chip or in a tube using a proteolytic enzyme such as trypsin.
When the product is finally purified by electrophoresis, the band of the desired protein is cut out from the gel, and the protein is decomposed in the gel by a proteolytic enzyme. A peptide fragment is extracted from there and peptide mapping is performed using a protein chip.

(B) Data analysis: The peak number derived from trypsin is removed, and the mass number of the detected peptide fragment is input to the protein identification software.

When the marker protein of the present invention is identified, the marker protein can be detected by a conventional method for detecting the protein. For example, the marker protein of the present invention can be detected by an analysis method utilizing an antigen-antibody reaction.

To carry out the antigen-antibody reaction, the marker protein of the present invention or a peptide having a partial amino acid sequence thereof is obtained and used as an immunogen to prepare an antibody that recognizes the marker protein of the present invention. The antibody may be a polyclonal antibody or a monoclonal antibody.

For example, a polyclonal antibody is obtained by immunizing a mammal with a marker protein as an antigen, collecting blood from the mammal, and separating the antibody from the collected blood.
It can be obtained by purification. The antigen can be used for immunization by dissolving it in a buffer containing an appropriate adjuvant, if desired. After the immunized mammal is bred for a certain period of time, a small amount of the blood of the mammal is sampled, the antibody titer is measured, and when the antibody titer rises, the antigen is administered an appropriate number of times depending on the situation. Blood is collected from an immunized mammal 1-2 months after the last administration by a conventional method, and the blood is centrifuged, for example,
A polyclonal antibody that recognizes the marker protein of the present invention can be obtained by separating and purifying by a conventional method such as precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange chromatography, affinity chromatography and the like. Obtainable.

The monoclonal antibody can be obtained by preparing a hybridoma by cell fusion of antibody-producing cells and a myeloma cell line, and culturing the hybridoma. The hybridoma can be produced by a known method (G. Kohler et al., Nature, 256 495 (1975)). The hybridoma obtained by cell fusion is cloned by a limiting dilution method or the like, and further screened by an enzyme immunoassay using the marker protein of the present invention to produce a monoclonal antibody that specifically recognizes the marker protein. Can be obtained. In order to produce the desired monoclonal antibody from the thus obtained hybridoma, the hybridoma may be cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody may be purified from the culture supernatant or ascites. . Purification of the monoclonal antibody from the culture supernatant or ascites can be carried out by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be appropriately combined and used.

The method for detecting or quantifying a marker protein present in human blood using the antibody obtained as described above is known to those skilled in the art. Specifically, RIA, E
IA, ELISA, immunocompetitive assay and the like can be used. The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

[0039]

[Example] (Method) 1. Samples Used in Examples Plasma was used before the treatment of a patient determined to be blood using the diagnostic criteria shown below and after administration of Keishibukuryogan. Keishibukuryogan is a typical drug used in blood patients. Plasma was frozen at −80 ° C. immediately after collection and stored in a frozen state until just before the test. As a control, plasma collected from two healthy adult males was used.

2. Method of diagnosing blood patients The selection of blood patients was carried out by the scores calculated according to the criteria for blood diagnosis by the research group of the Science and Technology Agency shown below.
The determination of the therapeutic effect after administration was also made based on the change in the score. At the same time, the hemodynamic parameters (Hemorheol
The measurement was also performed for the (ogical parameter).

[0041]

Criteria 20 points or less: non-blood condition, 21 points or more 40 points or more severe blood condition

As for the scores, those which were clearly recognized were given the corresponding scores, and those which were mild were given 1/2. Note 1) Rough skin, roughness, and rupture. Note 2) Expansion of capillaries, spider hemangiomas, etc.

3. The following items were measured as blood rheological parameters.・ Whole blood viscosity ・ Plasma viscosity ・ Erythrocyte deformability ・ DEA ・ Hematocrit value

4. Blood Diagnostic Marker-Detection Method Using Protein Chip A protein chip system manufactured by CIPHERGEN was used.
Hereinafter, a portion having a functional group immobilized on the protein chip will be referred to as a spot.

(Method 1) Method for detecting blood-specific peak in plasma using IMAC3 protein chip 5 μL of 50 mmol / L CuSO ₄ was added per spot,
Shake for 5 minutes at room temperature. The spots were washed with ultrapure water to remove unbound Cu ²⁺ . 5 per spot
A μL buffer (100 mmol / L Sodium acetate (pH4)) was added, and the mixture was shaken at room temperature for 5 minutes. The spots were washed with ultrapure water.

To each spot, 200 μL of buffer was added and shaking for 5 minutes was repeated twice to equilibrate the solid-phased functional groups. 100 μL of plasma with buffer 1 μL
Diluted and applied on the spot. After shaking at room temperature for about 30 minutes, the diluent was removed. 200 per spot
Non-specific binding was removed by adding μL of PBS and repeating shaking for 5 minutes three times. About 400 μL of ultrapure water was added per spot and the removal was repeated twice to remove the buffer.

After the spots were sufficiently dried, 0.5 μL of EAM was applied to each spot to crystallize the residual molecule. After the spot was dried, 0.5 μL of EAM was applied again. The protein chip was inserted into a protein chip reader (PBS-II), which is a TOF-MS measuring device, and the molecular weight spectrum of the protein remaining in the spot was analyzed.

(Method 2) Method for detecting blood-specific peak in plasma using SAX2 protein chip 200 μL buffer (50 mmol / L Sodiu per spot)
m acetate (pH 4.0) or 50 mmol / L Tris-HCl (pH 8.
(0)) was added and shaking for 5 minutes was repeated twice to equilibrate the immobilized functional groups. 1 μL of plasma with buffer 1
It was diluted to 00 μL and applied on the spot. At room temperature
After shaking for 30 minutes, the diluent was removed. 200 μL of buffer was added per spot and shaking for 5 minutes was repeated 3 times to remove nonspecific binding. About 4 per spot
The buffer was removed by adding 00 μL of ultrapure water and repeating removal twice.

After the spots were sufficiently dried, 0.5 μL of EAM was applied per spot to crystallize the residual molecule. After the spot was dried, 0.5 μL of EAM was applied again. The protein chip was inserted into a protein chip reader (PBS-II), which is a TOF-MS measuring device, and the molecular weight spectrum of the protein remaining in the spot was analyzed.

(Method 3) Method for detecting blood-specific peak in plasma using WCX2 protein chip 200 μL buffer (50 mmol / L Sodiu per spot)
m acetate (pH 4.0) or 50 mmol / L Ammonium acetate
(pH 6.0)) was added and shaking for 5 minutes was repeated twice to equilibrate the immobilized functional groups. 1 μL of plasma was diluted to 100 μL with a buffer and applied on a spot.
After shaking at room temperature for about 30 minutes, the diluent was removed. 200 μL of buffer was added per spot and shaking for 5 minutes was repeated 3 times to remove nonspecific binding. About 400 μL of ultrapure water was added per spot and the removal was repeated twice to remove the buffer.

After the spots were sufficiently dried, 0.5 μL of EAM was applied per spot to crystallize the residual molecule. After the spot was dried, 0.5 μL of EAM was applied again. The protein chip was inserted into a protein chip reader (PBS-II), which is a TOF-MS measuring device, and the molecular weight spectrum of the protein remaining in the spot was analyzed.

(Result)

[0054]

[Table 1]

[0055]

[Table 2]

(Result 1) The results of detection of blood-specific peaks in plasma using the IMAC3-Cu chip are shown in FIGS. No significant change in the peak pattern was observed between the two healthy subjects, but a distinct peak was detected when the healthy subjects and blood patients were compared. Healthy subjects and blood patients before drug administration
Comparison with Sample T shows m / z 8086 (peak 6), 9
251 (peak 7), 14929 (peak 9), 15087 (peak 10), 152
88 (peak 11), 15827 (peak 12), 28177 (peak 18), 28
900 (peak 19), 30514 (peak 20), 33368 (peak 21), 6
It was confirmed that the peaks observed in 0191 (peak 22) and 66606 (peak 23) are present in healthy humans, whereas they disappeared or were clearly reduced in Sample T. In addition, m / z 3865 (peak 1), 4547 (peak 5), 9910 (peak
8), 16992 (peak 13), 17183 (peak 14), 18293 (peak
15), 19835 (peak 16) and 20016 (peak 17).
A peak was observed that appeared only in ample T, or was clearly increased compared to healthy individuals. Regarding peaks 16 and 19, peak fluctuations toward the normal level were observed in Sample T after blood treatment with Keishibukuryogan. For these, it is considered that the peaks fluctuated as a result of the therapeutic effect of drug administration. Samp
Sample M based on the peak variation observed in le T
Was measured, peaks 1, 5, 6, 7, 8, 9, 10, 11,
At 12, 13, 14, 15, 16, 17, 21, and 23, Samp
The same variation as le T was confirmed. Furthermore, three peaks of m / z 3904 (peak 2), 3929 (peak 3), and 4411 (peak 4), which appeared or increased only in Sample M, were confirmed as compared with healthy persons. In Sample M, changes toward the normal level were observed at peaks 2, 3, 4 and 16 by blood treatment by administration of Keishibukuryogan. Of which the peak
In 16 samples, variation was observed in both Sample T and M. From the above, for diagnosing blood condition using IMAC3-Cu chip
23 kinds of peaks were detected.

(Result 2) The results of detection of blood-specific peaks in plasma using SAX2 protein chip (anion exchange chip) are shown in FIGS. 4 to 7 and Table 1. No significant change in the peak pattern was observed between the two healthy subjects, but a distinct peak was detected when the healthy subjects and blood patients were compared. Moreover, even if the same SAX2 protein chip was used, it was possible to confirm different peak patterns, that is, variations in vivo of different peptides, by varying the washing conditions. When the washing buffer was set to 50 mmol / L Sodium acetate (pH 4) and healthy humans and Sample T before drug administration were compared, m / z 3906
(Peak 25), 3918 (peak 26), 4124 (peak 27) and 28
The peak observed in 092 (peak 28) was confirmed in healthy individuals, whereas in Sample T, disappearance or a clear decrease was observed. Based on the peak fluctuations observed in Sample T, when Sample M was measured, peak 2
The same fluctuations as Sample T were confirmed in 5, 26 and 28. Peak 27 was higher than that of healthy subjects, but decreased as a result of blood treatment with Keishibukuryogan. As for the peak 28, the fluctuation toward the healthy subject level after the treatment was confirmed only in Sample M. Wash buffer
When 50 mmol / L Tris-HCl (pH 8) was compared with Sample T before administration of the drug in healthy subjects, m / z 3906 (peak 29), 40
62 (peak 30), 5445 (peak 31), 5601 (peak 32), 8520
The peaks observed in (Peak 34) and 8641 (Peak 35) were confirmed in healthy individuals, whereas in Sample T, disappearance or a clear decrease was observed. Also, m / z 9
136 (peak 36), 9299 (peak 37), 13687 (peak 38), 15
065 (Peak 39), 15857 (Peak 40) and 16015 (Peak 4)
In 1), we confirmed a peak that appeared only in Sample T or that was clearly increased compared to healthy people. Regarding peaks 34 and 35, peak fluctuations toward the normal level were observed in Sample T after blood treatment with Keishibukuryogan. When Sample M was measured based on the peak fluctuations observed in Sample T, the same fluctuations as in Sample T were confirmed at peaks 29, 30, 31, 32, 36, 37, 38, 39, 40, and 41. did. Moreover, m / z 8 increases only in Sample M compared to healthy subjects
One peak of 414 (peak 33) was confirmed. From the above, 17 kinds of peaks for diagnosing blood condition were detected using SAX2 chip.

(Result 3) The results of detection of blood-specific peaks in plasma using the WCX2 protein chip (cation exchange chip) are shown in FIGS. No significant change in the peak pattern was observed between the two healthy subjects, but a distinct peak was detected when the healthy subjects and blood patients were compared. Even if the same WCX2 protein chip was used, it was possible to confirm different peak patterns, that is, variations in different peptides in vivo, by varying the washing conditions. When the wash buffer was 50 mmol / L Sodium acetate (pH4) and the healthy human and sample T before drug administration were compared, m / z 4063
The peak observed in (peak 42) was confirmed in healthy individuals, whereas in Sample T, disappearance or a clear decrease was observed. Also, m / z 13713 (peak 46), 15065 (peak 47), 15815 (peak 48), 15889 (peak 49), 16033
In (Peak 50), 16086 (Peak 51) and 16221 (Peak 52), peaks that appeared only in Sample T or were clearly recognized to be increased as compared with those of normal subjects were confirmed. Regarding peaks 49, 51 and 52, peak fluctuations toward the normal level were observed in Sample T after blood treatment with Keishibukuryogan. When Sample M was measured based on the peak fluctuation observed in Sample T, peaks 42, 47, 48, 49, 50, 51 and 52 were
The same fluctuation as in Sample T was confirmed. In addition, m / z 4568 (peak 4
3), 9154 (peak 44) and 9310 (peak 45) were confirmed. Wash buffer at 50 mmol / L Ammonium aceta
As a te (pH 6), a comparison was made between healthy subjects and Sample T before drug administration, and m / z 4278 (peak 54), 4619 (peak 5
6), 6156 (peak 57), 6242 (peak 58), 7737 (peak 6)
0), 8111 (peak 63), 8573 (peak 64), 8782 (peak 6)
5), 8903 (peak 66), 9257 (peak 67), 9466 (peak 6)
8), 10231 (Peak 69) and 14917 (Peak 71), the peaks observed in healthy subjects were compared to Sample
At T, there was a disappearance or a clear decrease. Also, m
/ z 7531 (peak 59), 7988 (peak 61), 7940 (peak 6)
2), 12819 (peak 70), 15083 (peak 72), 15279 (peak
73), 15814 (Peak 74), 15885 (Peak 75) and 16042
At (peak 76), a peak that appeared only in Sample T or was clearly increased compared to a healthy person was confirmed. For peaks 62, 65 and 76,
In Sample T after blood treatment with Keishibukuryogan, peak fluctuation toward the level of healthy subjects was observed. Sample T
The sample M was measured based on the peak fluctuations observed in the sample, peaks 54, 56, 58, 59, 60, 61, 62, 6
3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75
In and 76, the same variation as Sample T was confirmed. Furthermore, two peaks of m / z 3901 (peak 53) and 4410 (peak 55) were confirmed, which were increased only in Sample M as compared with healthy subjects. In Sample M as well as Sample T,
Peak 62, 65, 75 due to blood treatment by administration of Keishibukuryogan
At 76 and 76, a change toward the normal level was observed. For peaks 53, 55, 61 and 74,
Only le M confirmed the fluctuation toward the normal level.
From the above, to diagnose the blood condition using the WCX2 chip
35 peaks were detected.

(Discussion) When the protein in plasma of a blood patient was comprehensively measured using a protein chip system, a plurality of peak patterns clearly different from those of a healthy person were shown. Table 3 shows hemodynamic parameters of T and M before and after blood treatment used as samples.

[0060]

[Table 3]

In both Samples T and M, an improvement in blood viscosity was observed along with an improvement in blood, and the result of this time is not only a blood marker but also a marker for detecting abnormal blood viscosity. Conceivable. As a result of the analysis, the peak patterns in Samples T and M were very similar. 75 of the peaks that show different fluctuations from normal
% Or more showed common behavior. These detected peaks appear to be extremely important markers for detecting patients with bloody symptoms.

Most of the blood symptoms do not occur suddenly, and it is considered that genetic factors of individuals and environmental factors such as lifestyle are intricately intertwined with each other. That is, it is considered that the occurrence of this symptom is not caused by one cause but is caused by a combination of a plurality of factors. Since the analysis patterns in the plasma of healthy humans used this time are very similar, the samples T and M
The difference in the peaks observed in 1. is more likely to be due to the difference in the cause of the onset of blood symptoms than the individual difference in the normal range. The peaks observed this time, which show different behaviors between samples T and M, are expected to be detected in other blood patients, and may be used as a criterion for estimating the patient background in the future. high.

[0063]

INDUSTRIAL APPLICABILITY According to the present invention, a marker protein useful for diagnosing blood has been identified, and by using this marker protein, diagnosing blood can be performed easily. Furthermore, according to the present invention, a marker protein whose expression changes before and after blood treatment is identified, and by using this marker protein, it becomes possible to facilitate the follow-up observation of blood treatment. In addition, this marker can also be used for patients with abnormal blood viscosity.

[Brief description of drawings]

FIG. 1 shows analysis results using an IMAC3-Cu chip.

FIG. 2 shows an analysis result using an IMAC3-Cu chip.

FIG. 3 shows analysis results using an IMAC3-Cu chip.

FIG. 4 shows the analysis results using a SAX2 protein chip (anion exchange chip / pH4).

FIG. 5 shows analysis results using a SAX2 protein chip (anion exchange chip / pH4).

FIG. 6 shows analysis results using a SAX2 protein chip (anion exchange chip / pH8).

FIG. 7 shows analysis results using a SAX2 protein chip (anion exchange chip / pH8).

FIG. 8 shows analysis results using a WCX2 protein chip (cation exchange chip / pH 4).

FIG. 9 shows the results of analysis using a WCX2 protein chip (cation exchange chip / pH 4).

FIG. 10 shows analysis results using a WCX2 protein chip (cation exchange chip / pH 6).

FIG. 11 shows the analysis results using a WCX2 protein chip (cation exchange chip / pH 6).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Koji Terazawa             463-5 Horikawacho, Toyama City, Toyama Prefecture (72) Inventor Ikuo Suiki             151-26 Hotarukawa, Fuchu-cho, Neguro-gun, Toyama Prefecture (72) Inventor Keiichi Koizumi             2-2 Minamitaikoyama, Kosugi Town, Imizu District, Toyama Prefecture             Medical and Pharmaceutical University Staff Dormitory 2-305 (72) Inventor Hirozo Goto             1-2-16 Momoicho, Toyama City, Toyama Prefecture F term (reference) 2G045 AA01 AA25 CA25 CA26 DA36                       FA34 FB03

Claims (5)

[Claims] 1. A copper ion-immobilized chip, an anion exchange chip and / or a cation exchange chip are allowed to react with a human body fluid, and the protein interacting with the chip is analyzed by mass spectrometry to obtain a patient and a healthy person. A method for diagnosing abnormal blood or blood viscosity, which comprises comparing analysis results. 2. (1) A copper ion-immobilized chip is allowed to react with human body fluid, and the protein interacting with the chip is analyzed by mass spectrometry, and 3865 (3861-3865), 3904, 39 at m / z.
29, 4411, 4547 (4542-4547), 8086, 9251 (9251-9277), 9
910 (9902-9910), 14929 (14919-14929), 15087 (15079)
-15087), 15288 (15288-15294), 15827 (15824-1582)
7), 16992, 17183, 18293, 19835 (19815-19839), 20
016, 28177 (28094-28194), 28900 (28856-28909), 305
14, 33368 (33274-33395), 60191, or 66606 (66507-
66620) or the presence or amount of any one or more of the proteins having, or (2) reacting an anion exchange chip with human body fluid and analyzing the protein interacting with the chip by mass spectrometry, m / z 3906 (3905-3906), 3918 (39
17-3921), 4124 (4119-4124), 28092 (28084-2817)
8), 4062 (4059-4062), 5445 (5444-5445), 5601, 8
414 (8404-8414), 8520 (8508-8520), 8641 (8632-865)
0), 9136 (9131-9142), 9299 (9288-9299), 13687 (1
3681-13707), 15065 (15039-15068), 15857 (15856-1)
5881) or 16015 (16015-16049) to detect the presence or amount of any one or more proteins, or
(3) A cation-exchange chip and a human body fluid are reacted, and the protein interacting with the chip is analyzed by mass spectrometry, which is 4063, 4568 (4564-4568), 9154 (9146-915) at m / z.
4), 9310 (9302-9311), 13713 (13699-13713), 15065
(15054-15075), 15815, 15889 (15879-15894), 1603
3, 16086, 16221, 3901, 4278, 4410 (4409-4410), 46
19, 6156 (6155-6156), 6242 (6242-6245), 7531 (75
21-7533), 7737 (7725-7746), 7988 (7898-7900), 7
940 (7940-7942), 8111 (8107-8111), 8573 (8567-85)
73), 8782 (8776-8782), 8903 (8902-8904), 9257
(9242-9263), 9466 (9465-9466), 10231 (10224-102)
31), 12819 (12801-12819), 14917, 15083 (15062-1508
4), 15279, 15814 (15814-15827), 15885 or 16042. A method for diagnosing blood or blood viscosity abnormality, which comprises detecting the presence or amount of any one or more proteins having 15885 or 16042. 3. The method according to claim 1, which is performed for observing the therapeutic effect of blood or blood viscosity abnormality. 4. A method for diagnosing a disease characterized by blood or blood viscosity abnormality and / or observing a therapeutic effect.
The method according to claim 1 or 2. 5. A disease characterized by abnormal blood or blood viscosity is cerebrovascular disease, coronary artery disease, chronic hepatitis, rheumatoid arthritis, Behcet's disease, menstrual disorders / infertility, menopause, nephrosclerosis, or peripheral diseases. The method according to claim 4, wherein the method is vascular disorder.