JPH116832A - Method and reagent for inspection of kawasaki disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a method and a reagent in which whether a patient suffers from Kawasaki disease or not can be inspected simply by measuring the concentration of a vessel endothelial cell growth factor/a vessel permeable factor (VEGF) in a human blood serum. SOLUTION: In a method, the concentration of a VEGF in a human blood serum is measured, and whether a patient suffers from Kawasaki disease or not is inspected. That is to say, the method is based on a relationship that the concentration of the VEGF displays a significantly high value in the case of Kawasaki disease as compared with an infectious chip patient and a healthy child. A method of measuring the concentration of the VEGF in the human blood serum is not limited especially, and, for example, a method using a substance which is specifically bonded to the VEGF can be enumerated. As the substance, e.g. an antibody or a receptor which is bonded to the VEGF can be enumerated. As an antibody with reference to the VEGF, a monoclonal antibody and a polyclonal antibody can be used. In addition, a specified antibody such as a human antibody or the like can be used. The antibody is used after it is labeled by, e.g. a red blood cell, a latex, a radioactive isotope or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for examining whether a patient has Kawasaki disease, a method for examining the medical condition of a patient with Kawasaki disease, and a reagent for these examinations.

[0002]

2. Description of the Related Art Kawasaki disease is a febrile eruptive disease that frequently occurs in children of 4 years or younger of unknown cause. The true nature of Kawasaki disease is systemic vasculitis characterized by coronary arteritis. Today, children with acquired Kawasaki disease have acquired coronary aneurysms due to thrombotic occlusion of the coronary aneurysm, sudden cardiac death due to thrombotic occlusion of the coronary aneurysm, or have a sequela of coronary aneurysm at a frequency of 10 to 20%. It is attracting attention as one of heart diseases. The diagnosis of Kawasaki disease is made symptomatically because there are no specific laboratory findings.
The main symptoms of Kawasaki disease are fever, which lasts more than 5 days,
Changes in the extremities of the extremities, irregular rashes, bilateral ocular mucosal hyperemia, flushing lipstick, erosive rash on the strawberry tongue, oropharyngeal mucosa, and non-suppurative cervical lymphadenopathy in the acute phase are known.

As a diagnosis of Kawasaki disease, at present, blood tests include measurement of the number of leukocytes, which is an acute inflammatory response, enhancement of erythrocyte sedimentation, CRP test, α2 globulin test, and platelet count. And white blood cell count, chest X-ray photography, electrocardiogram measurement, echo measurement, and the like. However, blood tests and urine tests are tests for inflammation in general, and are not necessarily established as tests for Kawasaki disease. In addition, X-ray photography, electrocardiogram measurement, and echo measurement are not simple methods that can be performed as tests for Kawasaki disease, because special equipment and devices are required. Although these tests are used to understand the pathology of Kawasaki disease, the test results do not always accurately reflect the pathology.

On the other hand, vascular endothelial growth factor / vascular pe
rmeability factor (VEGF) is secreted from mouse, rat, guinea pig, bovine and human normal or tumor cell lines, and is also found to be present in the brain, pituitary gland, kidney and ovary by tissue (Fe
rrara, N., et.al. Endocrine Reviews 13:18 (1992)).

Regarding the properties of human VEGF, angiogenesis and metastasis of breast cancer (Weider, N, et. Al. N. Engl. J. Med. 324: 1 (199
1)) and angiogenesis of renal cell carcinoma (Ayumi of Medicine, 168: 231 (199)
4)) or angiogenesis in retinal diseases (Adamis, AP et.a
l., Biochem. Biophys. Res. Comm., 193: 631 (1993)).

Further, cDNA of human VEGF has already been isolated, and its nucleotide sequence and deduced amino acid sequence have been specified. In addition, the human VEGF gene has four types of isoforms with different numbers of amino acid residues due to differences in splicing (the number of amino acid residues is 121, 165, 189, and 206)
Are produced, and 121 amino acid residues (V
EGF121) and 165 amino acid residues (VEGF165) are considered mature proteins (Ferrara, N., e.
t.al. Endocrine Reviews 13:18 (1992)). VEGF121 is VE
Although the carboxyl-terminal 44 amino acids of GF165 have been deleted, it is not clear whether there is a difference between VEGF121 and VEGF165 in their effects on vascular endothelial cells.

Among them, a monoclonal antibody against human VEGF121 has already been obtained by the present inventors (Japanese Patent Application No. 7-62841), and the monoclonal antibody and human V
Demonstrates that VEGF can be measured by enzyme immunoassay using polyclonal antibody against EGF121
(Japanese Patent Application No. 7-141271).

[0008]

The object of the present invention is to provide a method for easily examining whether a patient has Kawasaki disease, a method for easily examining the pathology of Kawasaki disease, and a reagent for these examinations. Make it an issue.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, have clarified that there is a close relationship between Kawasaki disease and VEGF concentration in human blood. As a result, they have found that it is possible to examine whether a patient has Kawasaki disease by measuring the VEGF concentration in human blood. Further, the present inventors have also elucidated that changes in blood VEGF concentration in Kawasaki disease reflect changes in the patient's disease state,
The present inventors have found that it is possible to examine a patient's condition by measuring a change in VEGF concentration, and to further predict the prognosis, thereby completing the present invention.

That is, the present invention provides (1) a method for examining Kawasaki disease characterized by measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor in human serum, and (2) vascular endothelial cell growth factor / blood vessel. The test method according to (1), wherein the concentration of vascular endothelial cell growth factor / vascular permeability factor in human serum is measured using a substance that specifically binds to the permeability factor, (3)
The test method according to (2), wherein the substance that specifically binds to vascular endothelial cell growth factor / vascular permeability factor is an antibody.
(4) an agent for testing Kawasaki disease, comprising as an active ingredient a substance that specifically binds to vascular endothelial cell growth factor / vascular permeability factor; (5) specific to vascular endothelial cell growth factor / vascular permeability factor The test agent according to (4), wherein the substance that specifically binds is an antibody.

[0011]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for examining the presence or absence of Kawasaki disease, which comprises measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF) in human serum. .

[0012] The present inventors have found a close relationship between Kawasaki disease and VEGF concentration, that is, the VEGF concentration is significantly higher in patients with Kawasaki disease than in patients with pediatric infections and healthy children. By measuring the serum VEGF concentration based on the relationship, it was possible to examine whether the patient's disease was Kawasaki disease.

The method for measuring the concentration of VEGF in human serum according to the present invention is not particularly limited, and examples thereof include a method using a substance that specifically binds to VEGF. Examples of such a substance include an antibody and a receptor that bind to VEGF. As an antibody against VEGF, a polyclonal antibody can be used in addition to a monoclonal antibody. It is also possible to use special antibodies such as humanized antibodies.

The antibody can be appropriately labeled and used. Labels include, for example, red blood cells, latex, radioisotopes,
Enzymes, luminescent materials, fluorescent materials, metal molecules, metal gels, bacteriophages, and the like can be used.

On the other hand, a known receptor for VEGF can be used instead of the antibody (Shibuya. M, et al.
Oncogene, 5,519 (1990), Jaime. C, et al. Science, 255, 98.
9 (1992)).

According to the present invention, the pathological condition of Kawasaki disease can be examined by measuring the change in the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF) in human serum. That is, in patients with Kawasaki disease, serum V
Although the EGF concentration showed a high value, the subacute stage, the VEGF concentration in serum decreased as the disease state toward remission to the convalescent phase, by measuring the change in serum VEGF concentration, for example, in patients with Kawasaki disease It is possible to examine changes in medical conditions, current medical conditions, and future medical conditions (patient prognosis). For example, if the serum VEGF concentration in the acute phase of Kawasaki disease patients who showed a high level of serum VEGF concentration subsequently shifted to a low level, the patient could be judged to be recovering, If the patient still shows a high value, it can be determined that the patient is still in an acute phase. Therefore, this change in VEGF concentration can be regarded as an index for determining the effect of treatment of Kawasaki disease.

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

[0018]

【Example】

[Example 1] Preparation of anti-vascular endothelial cell growth factor / vascular permeability factor (VEGF) polyclonal antibody Isolated human VEGF cDNA was produced in Escherichia coli as a fusion protein (GST-VEGF) with glutathione S-transferase (GST). A rabbit anti-VEGF polyclonal antibody was prepared according to a conventional method using the obtained protein as an antigen.

The serum of a rabbit having an increased antibody titer is separated,
Rabbit anti-ion by anion exchange column chromatography
An IgG fraction of VEGF polyclonal antibody was obtained (Kondo, S, et.
al.Biochemical and Biophysical Research Communicat
ion 19, 1234 (1993)).

Example 2 An enzyme-labeled anti-VEGF polyclonal antibody A part of the IgG fraction was digested with pepsin to prepare F (ab ') 2.
Hinge method (Ishikawa, et al. J. Immunoassay, 4,209 (198
3)) to bind to peroxidase (horseradish),
A rabbit anti-VEGF polyclonal antibody labeled with peroxidase was obtained.

Example 3 Measurement of Serum VEGF Concentration in Kawasaki Disease Patients Serum VEGF concentration in Kawasaki disease patients was measured by an enzyme immunoassay as shown below. Anti-VEGF polyclonal antibody
(5 μg / mL) was spread on a 96-well plate at 100 μl / well at a temperature of 4 ° C overnight, and then PB containing 0.1% bovine serum albumin (BSA).
Washed four times with S. 37 ° C with 1% BSA, 0.1 M sodium chloride, 0.1% sodium azide, 0.1 M sodium carbonate buffer (pH 6.5)
After blocking for 4 hours, 1% BSA, 0.4% gelatin, 1 mM
Contains magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1 M sodium chloride, 0.1% sodium azide
Standard VEGF dissolved in serum or the same sample diluent diluted with 50 mM sodium phosphate buffer (pH 7.0) (sample diluent)
And left at room temperature for 1 hour. After washing six times with PBS containing 0.1% BSA, a peroxidase-labeled anti-VEGF polyclonal antibody was added at 100 μl / well, and the mixture was reacted at room temperature for 1 hour. Again, after washing eight times with PBS containing 0.1% BSA, 0.125% (w / v) orthophenylenediamine, 0.015% hydrogen peroxide, 0.2 M tris (hydroxymethyl (aminomethane) -citrate buffer (pH 5.2 )
100 μl / well was added thereto and reacted at room temperature for 30 minutes. 2N sulfuric acid was added at 100 μl / well, and after stopping the reaction, 650 nm
The absorbance at 490 nm against the absorbance of
Vmax, manufactured by Molecular Devices).

Example 4 Measurement of Serum VEGF Concentration in Kawasaki Disease Patients 19 Kawasaki disease patients, 17 pediatric infectious disease patients, and healthy children
The serum VEGF level was measured in 46 cases. The distribution, average value, and range of each measurement value are shown in FIG. As a result, Kawasaki disease patients showed significantly higher values than pediatric infectious disease patients and healthy children.

Further, in patients with Kawasaki disease, the VEGF levels in the serum during the acute, subacute, and convalescent phases were measured, and changes at the individual level are shown in FIG. The serum VEGF level in the acute phase is significantly higher than the serum VEGF level in the subacute phase and the convalescent phase, and the serum level of serum VEGF increases from the acute phase to the subacute phase and the convalescent phase. VEGF levels had decreased.

[0024]

According to the present invention, there are provided a method for examining whether a patient has Kawasaki disease, a method for examining the medical condition of a patient with Kawasaki disease, and a reagent used for these tests.
According to the present invention, whether or not a patient has Kawasaki disease can be easily examined by measuring the concentration of VEGF in the serum of the patient. Further, by measuring a change in the VEGF concentration in the serum of a Kawasaki disease patient, the condition of the patient (for example, whether the patient is recovering or deteriorating) can be easily examined. .

[Brief description of the drawings]

FIG. 1 shows serum VEGF levels in Kawasaki disease.

FIG. 2 shows changes in serum VEGF levels in patients with Kawasaki disease.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Katsuhiko Matsuo, 2nd Okubo Tsukuba, Ibaraki Pref., Within Tsukuba Research Laboratories (72) Inventor Isamu Matsubara 2nd Okubo, Tsukuba, Ibaraki Pref., Tsukuba Research Laboratories, Ltd.

Claims (5)

[Claims] 1. A method for testing Kawasaki disease, comprising measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor in human serum. 2. The method according to claim 1, wherein the concentration of vascular endothelial cell growth factor / vascular permeability factor in human serum is measured using a substance that specifically binds to vascular endothelial cell growth factor / vascular permeability factor. Inspection methods. 3. The test method according to claim 2, wherein the substance that specifically binds to vascular endothelial cell growth factor / vascular permeability factor is an antibody. 4. An agent for testing Kawasaki disease, comprising a substance that specifically binds to vascular endothelial cell growth factor / vascular permeability factor as an active ingredient. 5. The test agent according to claim 4, wherein the substance that specifically binds to vascular endothelial cell growth factor / vascular permeability factor is an antibody.