JPH10300754A - Testing method and testing drug for acute hepatitis and fulminant hepatitis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To test whether a patient suffers from acute hepatitis or fulminant hepatitis with ease and to test the symptoms of a patient suffering from acute hepatitis or fulminant hepatitis with ease. SOLUTION: As acute hepatitis and fulminant hepatitis are closely related with the concentration of VEGF(vascular endothelial cell growth factor/vascular permeability factor) in human blood, it is possible to test whether a patient suffers from acute hepatitis or fulminant hepatitis failure with ease by measuring the concentration of VEGF in human blood. In addition, as the changes in the concentration of VEGF in the blood of patients suffering from acute hepatitis and fulminant hepatitis reflect the symptoms of the patients, it is possible to test the symptoms of the patients by measuring the changes in the concentration of VEGF in the blood of the patients.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for examining whether a patient has acute hepatitis or fulminant hepatitis, a method for examining the condition of an acute hepatitis patient or a fulminant hepatitis patient, and a reagent for these examinations. .

[0002]

BACKGROUND ART The annual incidence of acute hepatitis in Japan is about
It is estimated to be 350,000, of which about 1% has fulminant hepatitis, and the survival rate is about 30%. Acute hepatitis generally refers to acute hepatitis caused by the hepatitis virus, and hepatitis A virus (HAV) and hepatitis B virus (HB
V), hepatitis C virus (HCV) and the like.

As a test for acute hepatitis, blood biochemical tests include glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), and general blood tests include white blood cell count and platelet count.
In addition, a prothrombin time / hepaplastin test is used as a blood coagulation test, and HAV, HBV, and HCV-related markers are used as a virus test. However, these tests are tests related to all liver functions and have not been established as tests for acute hepatitis. In addition, acute hepatitis may cause fulminant or chronic disease, so it is necessary to accurately understand the patient's condition and prognosis, and the above tests are used for that purpose. The results may not accurately reflect the condition.

[0004] Fulminant hepatitis is considered to be a condition in which liver function has been significantly reduced due to rapid and widespread necrosis of hepatocytes. GOT of blood biochemistry test for fulminant hepatitis test
-GPT, total bilirubin, prothrombin time as a blood coagulation test, abdominal ultrasound, abdominal CT as an image diagnosis, and EEG as a test for hepatic encephalopathy. However, GOT, GPT, total bilirubin, and prothrombin time in blood biochemical tests are tests that determine the degree of hepatocellular damage, and therefore cannot always predict the prognosis of patients. It is not a simple inspection method because it requires facilities and equipment.

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)).

[0006] Regarding the characteristics 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. 7-162841), 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).

[0009]

DISCLOSURE OF THE INVENTION The present invention provides a method for easily examining whether a patient has acute hepatitis or fulminant hepatitis, a method for easily examining the condition of an acute hepatitis patient or a fulminant hepatitis patient, It is an object to provide a reagent for inspection.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, elucidated the close relationship between acute hepatitis and fulminant hepatitis and VEGF concentration in human blood,
By measuring the concentration of VEGF in human blood, we have found that it is possible to test whether a patient has acute or fulminant hepatitis. Furthermore, the present inventors have also elucidated that changes in the blood VEGF concentration in patients with acute hepatitis and fulminant hepatitis reflect changes in the pathology of the patients,
The present inventors have found that it is possible to examine a patient's condition by measuring a change in VEGF concentration in the patient's blood and to further predict the prognosis, thereby completing the present invention.

That is, the present invention provides (1) a method for detecting the presence or condition of acute hepatitis or fulminant hepatitis, which comprises measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor in human serum; Measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor in human serum using a substance that specifically binds to vascular endothelial cell growth factor / vascular permeability factor;
The test method according to (1), (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) vascular endothelial cell proliferation An agent for detecting the presence or condition of acute hepatitis or fulminant hepatitis, which comprises a substance that specifically binds to a factor / vascular permeability factor as an active ingredient; (5) a vascular endothelial cell growth factor / vascular permeability factor; The test agent according to (4), wherein the substance that specifically binds is an antibody.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for measuring the presence of acute hepatitis or fulminant hepatitis, which comprises measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF) in human serum. How to inspect.

The present inventors have found a close relationship between acute hepatitis or fulminant hepatitis and VEGF concentration, that is, in patients with acute hepatitis, VEGF is compared with healthy persons and patients with other liver diseases.
By measuring the serum VEGF concentration based on the relationship that the concentration shows a significantly high value, while fulminant hepatitis patients show significantly lower values compared to healthy subjects and other liver disease patients,
It became possible to test whether the patient's disease was acute hepatitis or fulminant hepatitis.

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)).

Further, according to the present invention, the pathology of an acute hepatitis patient or a fulminant hepatitis patient is examined by measuring a change in the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF) in human serum. You can also. That is, in patients with acute hepatitis, the serum VEGF concentration in the acute phase of acute hepatitis showed a high value, but in the recovery period, the serum VEGF concentration shows a low value, so the change in serum VEGF concentration should be measured. Due to, for example, changes in the condition of acute hepatitis patients, current conditions,
And it is possible to examine future conditions (prognosis of the patient). For example, if the serum VEGF concentration of an acute hepatitis patient whose serum VEGF concentration showed a high value subsequently shifts to a low value, the patient can be determined to be in a convalescent phase, while the patient still has a high VEGF concentration. If so, the patient can be determined to be in an acute phase. Therefore, this change in VEGF concentration can be used as an index for determining the effect of treating acute hepatitis.

Serum VEGF levels in patients with fulminant hepatitis who died and survived during the course of treatment were measured. In surviving cases, serum VEGF levels increased during the course of treatment. Showed a decrease in serum VEGF concentration. Therefore, by measuring the VEGF concentration in serum, it is possible to examine changes in the condition of fulminant hepatitis patients, current conditions, and future conditions (prognosis of patients). That is, if the serum VEGF concentration of a fulminant hepatitis patient gradually shifts to higher, it can be determined that the patient's condition is recovering, whereas if the serum VEGF concentration of the patient shifts to a lower level, Thus, it can be determined that the condition of the patient has deteriorated. Therefore, this change in VEGF concentration can be taken as an index for determining the effect of treatment for fulminant hepatitis.

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

[0020]

【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 VEGF in Serum of Patients with Various Liver Diseases
Measurement of concentration VEGF concentration in serum of various liver disease patients as shown below,
It was measured by enzyme immunoassay. Spread 100 μl / well of anti-VEGF polyclonal antibody (5 μg / mL) into a 96-well plate.
After being left overnight at 0 ° C, the plate was washed four times with PBS containing 0.1% bovine serum albumin (BSA). 1% BSA, 0.1M sodium chloride, 0.1%
With sodium azide, 0.1 M sodium carbonate buffer (pH 6.5)
After blocking at 37 ° C. for 4 hours, a 50 mM sodium phosphate buffer solution (pH 7.0) containing 1% BSA, 0.4% gelatin, 1 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1 M sodium chloride, and 0.1% sodium azide was used. ) (Sample diluent), dissolved standard VEGF was added to the serum diluted 3 or the sample diluent, and the mixture was allowed to stand at room temperature for 1 hour. PB with 0.1% BSA
After washing with S six times, a peroxidase-labeled anti-VEGF polyclonal antibody was added at 100 μl / well and reacted at room temperature for 1 hour. After washing again 8 times with PBS containing 0.1% BSA, 0.125% (w / v) orthophenylenediamine, 0.015% hydrogen peroxide, 0.2M tris
(Hydroxymethyl (aminomethane) -citrate buffer (pH
5.2) was added at 100 μl / well, and the mixture was reacted at room temperature for 30 minutes. After 2N sulfuric acid was added at 100 μl / well to stop the reaction, the absorbance at 490 nm relative to the absorbance at 650 nm was measured with a plate reader (M-Vmax, manufactured by Molecular Devices).

Example 4 VEGF in Serum of Patients with Various Liver Diseases
Measurement of concentration 21 patients with acute hepatitis, 40 patients with chronic viral hepatitis, 16 patients with fulminant hepatitis, 10 patients with primary biliary hepatitis, 12 patients with autoimmune hepatitis
The serum VEGF concentration was measured for each of the cases and 120 healthy subjects, and the average value and standard deviation value of the measured values were calculated. As a result, acute hepatitis patients showed significantly higher values compared to healthy subjects and others (Table 1 and FIG. 1).

[0025]

[Table 1] In patients with acute hepatitis, serum VEGF concentrations in the acute and convalescent phases were measured, and changes at the individual level were analyzed (FIG. 2). As a result, the serum VEGF concentration in the acute phase was significantly higher than the serum VEGF concentration in the recovery phase,
In each case, the serum VEGF concentration during the recovery period was lower than in the acute period.

On the other hand, fulminant hepatitis patients showed significantly lower values compared to healthy subjects and patients with other liver diseases (Table 1 and FIG.
1). In patients with fulminant hepatitis, serum VEGF levels were measured during the course of treatment in cases of death and survival, and changes at the individual level were analyzed (FIG. 3). As a result, in the surviving cases, the serum VEGF concentration was increased during the course of treatment, but in the dead cases, the serum VEGF concentration was decreased.

[0027]

According to the present invention, a method for examining whether a patient has acute hepatitis or fulminant hepatitis, a method for examining the condition of an acute hepatitis patient or a fulminant hepatitis patient, and a reagent used for these examinations are provided. . According to the present invention, whether or not a patient has acute hepatitis or fulminant hepatitis can be easily examined by measuring the concentration of VEGF in the serum of the patient. In addition, serum in patients with acute or fulminant hepatitis
By measuring the change in VEGF concentration, the patient's condition (for example, whether the patient is recovering or deteriorating) can be easily examined.

[Brief description of the drawings]

FIG. 1 shows serum VEGF concentrations in various liver disease patients.

FIG. 2 shows serum VEGF levels in acute and convalescent phases of acute hepatitis patients.

FIG. 3 shows changes in serum VEGF concentration in surviving and dead cases of fulminant hepatitis patients.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Isamu Matsubara 2nd Okubo Tsukuba, Ibaraki Pref.

Claims (5)

[Claims] 1. A method for determining the presence or condition of acute hepatitis or fulminant hepatitis, 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 for the presence or absence of acute hepatitis or fulminant hepatitis, which comprises 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.