JPH0933521A - Method and medicament for inspecting inflammatory disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize an effective inspection for inflammatory disease by measuring the quantity of VPF(Vascular Penetration Factor) in serum using labeled immunoassay. SOLUTION: A method for predicting the presence of an inflammatory disease, e.g. uncerative colitis, clonal disease or other inflammatory diseases, especially a method employing a monoclonal antibody or polyclonal antibody for VPF, can be carried out easily without requiring any special facility or experience. It is especially useful for mass health examination where a large quantity of specimens are examined at once. Furthermore, since a significant difference appears in the quantity of VPF in serum between the active interval and inactive interval of uncerative colitis or clonal disease, the inventive method is useful for diagnosing the active interval of both diseases, deciding the effect of treatment, and monitoring after operation.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a vascular permeability factor or vascular endothelial growth factor present in serum.
The abundance of a factor referred to as r) (hereinafter abbreviated as VPF) is measured, and based on the measured amount, ulcerative colitis is given to the serum donor.
The present invention relates to a method for predicting whether Crohn's disease or other inflammatory diseases are present, a method for providing information that is useful for a doctor in diagnosing an inflammatory disease, and inflammation. The present invention relates to a diagnostic agent for diagnosing inflammatory diseases, which is useful for diagnosing sexual disorders, and is used in the pharmaceutical industry.

[0002]

BACKGROUND OF THE INVENTION Ulcerative colitis is a non-specific inflammatory bowel disease, which is a difficult disease that often undergoes relapsing-remitting remission and is rarely completely cured. In addition, it has been reported that ulcerative colitis 10 years or more after onset develops colon cancer at a high rate (Tomoyuki Sato, a case of rectal cancer associated with ulcerative colitis and ulcerative disease in Japan). Examination of cases of cancer of colitis,
Journal of the Japanese Society for Colorectal Disease 45 , 868, 1992). Currently, endoscopic examination, enema examination, and biopsy tissue diagnosis are used to diagnose active ulcerative colitis, but it is easy to perform because special equipment and skilled technology are required. It is a method that cannot be done. In addition, the clone (Croh
n) It is a refractory inflammatory bowel disease that rarely completely cures, and it is thought that it is possible to suppress the transition to cancer by detecting it early and treating it early as in the case of ulcerative colitis. To be In particular, the diagnosis of active phase or non-active phase is important for determining the treatment method, determining the therapeutic effect and the recurrence / recurrence, and information for that is strongly required. C-reactive protein (CR
P) and the like, and CRP is widely used clinically as a diagnostic marker for inflammation because its concentration increases in serum of patients with inflammatory diseases.

On the other hand, the phenomenon of angiogenesis, that is, the proliferation, migration and invasion of capillary endothelial cells into tissues plays an important role in physiological or pathological phenomena such as fetal growth, wound healing, and cancer cell proliferation. [(Folkman, J., Cancer Res. 46: 467 (1986)]] As a factor that induces angiogenesis, basic fibroblast proliferation is a substance that directly acts on vascular endothelial cells. Factor (basic fib
roblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), vascular endothelial growth factor / vascular permeability factor (vascular endothelia)
l growth factor / vascular permeability factor, VP
F), platelet-derived e factor
ndothelial cell growth factor (PD-ECGF), etc. is also a substance that indirectly acts on vascular endothelial cells.
ng growth factor-α (TGF-α), transforming growth f
actor-β (TGF-β), angiogenin, tumor necrosis facto
r-α (TNF-α) etc. have been found [Folkman, J. & Sh
ing, Y., J. Biol. Chem., 267: 10931 (1992)].

Among these, VPF is secreted in mouse, rat, guinea pig, bovine and human normal or tumor cell lines, and depending on the tissue, it may be present in brain, pituitary gland, kidney and ovary. Has been revealed
[(Ferrara, N., et.al. EndocrineReviews 13:18 (199
2)]. Human VPF is also involved in breast cancer angiogenesis and metastasis [Weider,
N. et.al. N.Engl.J.Med. 324: 1 (1991)] and angiogenesis of renal cell carcinoma [Ayumi of Medicine, 168: 231 (1994)], or angiogenesis in retinal diseases [Adamis, AP et.al., Biochem.Bio
phys.Res.Comm., 193: 631 (1993)].

CDNA of the human VPF gene
Has already been isolated, its nucleotide sequence has been determined, and its amino acid sequence has been deduced. 4 kinds of proteins with different number of amino acid residues (121 amino acid residues, 165
, 189, and 206) were produced, and among them, those with 121 amino acid residues (VPF121) and 1
It is said that a protein having 65 amino acid residues (VPF165) is a mature protein [(Ferrara, N., et.al. Endocri
ne Reviews 13:18 (1992)]. VPF121 lacks the 44 amino acids at the carboxyl terminus of VPF165, but it is not clear whether VPF121 and VPF165 have a different action on vascular endothelial cells.

The present inventors have already obtained this monoclonal antibody against human VPF121, and it has been clarified that VPF can be measured by an enzyme immunoassay using the monoclonal antibody and the polyclonal antibody against human VPF121 (Japanese Patent No. : Peptides and monoclonal antibodies, filing date Jun. 1994
Day). In addition, the method for detecting VPF in the enzyme immunoassay has already been established by the present inventors, and a few pg
It has been revealed that VPF of 1 / ml can be detected (Japanese Patent Application No. 7-141271).

[0007]

DISCLOSURE OF THE INVENTION Based on the above findings, the present inventors find it useful as a diagnostic marker for inflammatory diseases, particularly for the diagnosis of ulcerative colitis and Crohn's disease. We have conducted intensive studies to find a diagnostic marker for inflammatory diseases.

[0008]

[Means for Solving the Problems] The present inventors have used sera from patients with ulcerative colitis and Crohn's disease as samples,
It was found that the VPF is useful as a diagnostic marker for inflammatory diseases such as ulcerative colitis and Crohn's disease by measuring the VPF concentration by a chemiluminescent enzyme immunoassay for VPF that has already been constructed. It was completed. That is, the present invention relates to an invention relating to a method for testing an inflammatory disease characterized by being based on the amount of a vascular permeability factor present in human serum and an inflammatory property characterized by comprising an antibody of a vascular permeability factor It consists of an invention relating to a disease test drug.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION VPF is known as well as its function as described above, and a known method can be used as a method for confirming the presence of VPF in serum. The preferred method for the present invention is VPF. Antigen-antibody reaction using a polyclonal antibody or a monoclonal antibody, particularly a label immunoassay, that is, a label using erythrocyte, latex, radioisotope, enzyme, luminescent substance, fluorescent substance, metal molecule, metal gel, bacteriophage, etc. as a label Immunoassay methods are preferred. In particular, it is preferable to use the chemiluminescence-detecting enzyme immunoassay method previously filed by the present inventors, which makes it possible to measure VPF in serum with high sensitivity and diagnose inflammatory diseases. In particular, the present invention becomes particularly useful. The chemiluminescence detection system enzyme immunoassay is a method of measuring light generated by using a luminescent substrate as a substrate of an enzyme, and uses an antibody against VPF, and the enzyme immunoassay is a monoclonal antibody against human VPF. A sandwich method or the like using an antibody or a polyclonal antibody can be applied.

[0010]

According to the clarification by the present inventors, by measuring VPF in human serum, inflammatory bowel disease such as ulcerative colitis or Crohn's disease or other inflammatory diseases can be detected. Method of predicting existence, especially VP
The method using a monoclonal antibody or polyclonal antibody against F does not require special equipment or experience and is simpler than the methods such as endoscopy, enema imaging, and biopsy tissue diagnosis that have been used up to now. Therefore, it is particularly useful when a large number of specimens are to be tested at one time, such as in mass screening. In addition, the active phase (severe symptoms) and inactive phase of ulcerative colitis or Crohn's disease are also useful. Since there is a significant difference in the serum VPF amount between (the symptoms are mild), it is useful for the diagnosis of the active phase in both diseases, the determination of the therapeutic effect, and the postoperative monitoring. Furthermore, since the amount of VPF in serum is highly correlated with the amount of CRP that is widely used as a diagnostic marker for inflammation, it is effective in diagnosing and treating many inflammatory diseases other than ulcerative colitis or Crohn's disease. It can also be widely used for determination of, post-operative monitoring, and the like.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below based on embodiments. (1) Preparation of anti-VPF polyclonal antibody The isolated human VPF cDNA was produced in Escherichia coli as a fusion protein (GST-VPF) with glutathione S-transferase (GST), and the obtained protein was used as an antigen to induce rabbit anti-prevention according to a conventional method. A VPF polyclonal antibody was prepared. The serum of the rabbit with an increased antibody titer was separated, and an IgG fraction of a rabbit anti-VPF polyclonal antibody was obtained by anion exchange column chromatography.

(2) Enzyme Labeling of Anti-VPF Polyclonal Antibody A part of the IgG fraction was digested with pepsin to prepare F (ab ') 2, which was then combined with alkaline phosphatase (derived from bovine small intestine) by the hinge method to give an alkaline solution. A rabbit anti-VPF polyclonal antibody labeled with phosphatase was obtained.

(3) Measurement of VPF Concentration in Serum of Patients with Ulcerative Colitis and Crohn's Disease Serum samples of 61 patients with ulcerative colitis (42 cases of active phase, 19 cases of inactive phase), serum samples of patients with Crohn's disease ( 28 active periods,
The amount of VPF in serum was measured using 17 cases of non-active period.

The measurement was carried out by the enzyme immunoassay method using the chemiluminescence detection method as shown below. 100 μl / well of anti-VPF polyclonal antibody (10 μg / ml) was spread on a 96-well plate and left overnight at 4 ° C., and then washed 4 times with 0.1% bovine serum albumin (BSA) and PBS. 1% B
After blocking with SA, 0.1M sodium chloride, 0.1% sodium azide, 0.2M sodium carbonate buffer pH 9.5 (1 hour at room temperature), 1% bovine serum albumin,
Diluted 10 times with 50 mM sodium phosphate buffer pH 7.0 (specimen diluent) containing 0.4% gelatin, 1 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1 M sodium chloride, and 0.1% sodium azide. The standard serum or the standard VPF dissolved in the diluted solution of the same sample was added and left at room temperature for 1 hour. 0.05% Tween 20,
After washing four times with Tris buffer pH 7.4 (TBS-T) containing 0.14 M sodium chloride and 5 mM potassium chloride, 100 μl / well of alkaline phosphatase-labeled anti-VPF polyclonal antibody was added and reacted at room temperature for 1 hour.
4 times with TBS-T, then 0.14M sodium chloride,
After being washed twice with Tris buffer pH 7.4 containing 5 mM potassium chloride, 0.1 M diethanolamine buffer p containing 1 mM magnesium chloride and 0.02% sodium azide
Lumiphos 530 (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 5 times with H10.0 was added at 100 μl / well, and the mixture was incubated at 37 ° C. for 30 minutes.
After standing for a minute, the luminescence intensity was measured with a plate luminometer (Luminus CT-9000D, manufactured by Diatron). The measurement results were classified into healthy subjects, patients with ulcerative colitis and patients with Crohn's disease, and the results displayed as a scatter plot are shown in FIG. 1. Compared with the sera of healthy subjects, the sera of patients with ulcerative colitis in the inactive stage, and the sera of patients with Crohn's disease, the VPF levels in the sera of the patients with active stage ulcerative colitis and the patients with Crohn's disease were significantly higher. Furthermore, for each of ulcerative colitis and Crohn's disease, the results of measuring the amount of serum VPF in the active phase and inactive phase for each patient are shown in FIG.
In most patients except one, the amount of VPF decreased in the non-active period compared to the active period. From these, it was revealed that the ulcerative colitis and Crohn's disease can be diagnosed by measuring the amount of VPF in serum. Furthermore, it was also found that the VPF amount decreases when the active phase shifts to the non-active phase, and thus it is highly correlated with the progression of the pathological condition.

Further, in order to clarify the correlation with CRP which is widely used clinically as an inflammation marker, VP for the transition of patient's pathological condition (time-dependent change).
The results of examining the relationship between the F amount and the CRP amount are shown in FIG. 3, and all 6 patients showed a high correlation between the VPF amount and the CRP amount. Therefore, it was revealed that various inflammatory diseases can be diagnosed by measuring the amount of VPF in serum.

[0016]

From the above, by measuring the amount of VPF in serum using the labeled immunoassay, it is possible to diagnose ulcerative colitis and Crohn's disease, and to judge the progress of disease state and therapeutic effect. I knew I could do it. Furthermore, it is considered that VPF in serum can be widely used as a diagnostic marker for various inflammatory diseases.

[0017]

[Brief description of drawings]

[Fig. 1] By enzyme immunoassay using chemiluminescence detection system, serum samples of ulcerative colitis patients (including those in active phase and inactive phase), serum samples of Crohn's disease patients (in active phase and inactive phase) The results of measurement of the amount of VPF in serum of the serum samples of healthy subjects and healthy subjects are classified into healthy subjects, patients with ulcerative colitis and patients with Crohn's disease, and displayed as scatter plots.

[Fig. 2] 13 patients with ulcerative colitis, 14 patients with Crohn's disease
Serum VPF levels in active and inactive periods were measured by enzyme-linked immunosorbent assay using chemiluminescence detection system, showing changes in serum VPF levels in active and inactive stages of each patient. is there.

FIG. 3 is a diagram showing changes over time in serum VPF amount and CRP amount in 6 patients with ulcerative colitis or Crohn's disease. The white circles indicate the CRP amount and the black circles indicate the VPF amount.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Iwao Omori Iku Omori, Tsukuba City, Ibaraki Prefecture 2nd Okubo Synthetic Co., Ltd. Tsukuba Research Institute (72) Inventor Katsuhiko Matsuo 2 Okubo, Tsukuba City Ibaraki Toagosei Co. Ltd. Tsukuba Synthetic Co., Ltd.

Claims (2)

[Claims] 1. A method for examining an inflammatory disease, which is based on the amount of a vascular permeability factor present in human serum. 2. An inflammatory disease test agent comprising an antibody of a vascular permeability factor.