JPH1048219A - Method and reagent for inspecting effect of treatment of liver cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To monitor transition from cirrhotis to liver cancer by employing the serum of a liver cancer patient as a sample and measuring the hemangioendocell growth factor/vessel penetration factor(VPF) in the serum by enzyme immunoassay. SOLUTION: A labeled immunoassay employing a substance reacting specifically on VPF, e.g. an anti-VPF antibody or VPF receptor, is suitable for measurement of VPF concentration in a serum. When an anti-VPF antibody is employed, for example, red blood cell, latex, radioisotope, enzyme, luminescent substance, fluorescent substance, metal molecule, metal gel or bacteriophage is employed as a label. In the enzyme immunoassay, antigen-antibody reaction is traced using the enzyme activity as an index and the quantity of antigen or antibody is measured. When the quantity of VPF in the serum is measured by labeled immunoassay, liver cancer can be inspected and transition from cirrhotis to liver cancer can be monitored.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to serum endothelial growth factor / vascular permeability factor.
factor / vascular permeability factor, below VEGF
/ VPF or VPF), and a method for testing liver cancer and a test drug used therefor. The test for liver cancer according to the present invention is used as a clinical test method for monitoring the transition from liver cirrhosis, which is a precancerous lesion, to liver cancer, and relates to a medical diagnosis technique and a test drug technique.

[0002]

2. Description of the Related Art It is known that many liver diseases eventually become cirrhosis. In addition, the rate of liver cancer complications at the time of death in cirrhosis cases is more than 80%, and it has been revealed that the prognosis of cirrhosis often depends on the presence or absence of carcinogenesis. Volume 171 Issue 14
p.1093-1096 (1994). Therefore, monitoring the transition from cirrhosis to liver cancer is clinically very important for determining the prognosis and determining the treatment method.
At present, the risk factors for carcinogenesis in cirrhosis cases are slightly different between type B cirrhosis and type C cirrhosis.
The serum bilirubin and pigment excretion test (ICG) 15-minute value, the latter, the age, the α-fetoprotein (AFP) value, and the history of heavy drinking are known. However, these factors have not actually been used as prognostic factors.

On the other hand, angiogenesis, that is, proliferation, migration and infiltration of tissue of capillary endothelial cells may play an important role in physiological or pathological phenomena such as fetal growth, wound healing, and proliferation of cancer cells. Known [(Folkma
n J., Cancer Res. 46: 467 (1986)]. Factors that induce angiogenesis include basic fibroblast growth factor (basic fibroblast growth factor) as a substance that directly acts on vascular endothelial cells.
owth factor, bFGF), acidic fibroblast growth factor (acidic
fibroblast growth factor, aFGF, vascular endothelial growth factor
actor / vascular permeability factor, VEGF / VPF), platelet-derived endothel
ial cell growth factor (PD-ECGF), etc.
wth factor-α (TGF-α), transforming growth factor-
β (TGF-β), angiogenin, tumor necrosis factor-α (T
NF-α) etc. [Folkman J. & Shing
Y., J. Biol. Chem., 267: 10931 (1992)].

[0004] VPF is secreted from mouse, rat, guinea pig, bovine and human normal or tumor cell lines, and has been shown to be present in the brain, pituitary gland, kidney and ovary by tissue. [(Ferrara N. et. Al.,
Endocrine Reviews 13:18 (1992)]. Human VPF is also used for angiogenesis and metastasis of breast cancer [Weider N. et. Al., N. Engl.
Med. 324: 1 (1991)], angiogenesis of renal cell carcinoma [Ayumi Ayumi, 168: 231 (1994)], or angiogenesis in retinal diseases [Adamis AP et. Al. Biochem. Biophys. Res. Com.
m. 193: 631 (1993)].

For the human VPF gene, its cDNA
Has already been isolated, its nucleotide sequence has been determined, and its amino acid sequence has been deduced. This gene is composed of four different proteins (one amino acid
21, 165, 189, 206), of which 121 amino acid residues
(VPF ₁₂₁ ) and 165 amino acid residues (VPF ₁₂₁ )
₁₆₅ ) is said to be a secreted protein [(Ferrara N. et.
al., Endocrine Reviews 13:18 (1992)]. VPF ₁₂₁ are those 44 amino acids of the carboxyl terminus of VPF ₁₆₅ is deficient, but during the VPF ₁₂₁ and VPF _165, not clear whether there is a difference in the effect on vascular endothelial cells.

Furthermore, it is acquired by the monoclonal antibodies already present inventors to human VPF ₁₂₁
(Japanese Patent Application No. 6-152805, "Peptides and monoclonal antibodies"). Furthermore, the monoclonal antibody and human V
By an enzyme immunoassay using a polyclonal antibody against PF _121, VPF number pg / ml has also been revealed that measurable (Japanese Patent Application No. 7-141271, "method of measuring vascular permeability factor").

[0007]

Under the circumstances described above, the present inventors cannot monitor the transition from cirrhosis to liver cancer by measuring the amount of VPF in the serum of liver cancer patients. It was considered. That is, an object of the present invention is to provide a method for monitoring the transition from cirrhosis to liver cancer.

[0008]

Means for Solving the Problems The present inventors used serum of a liver cancer patient as a sample, and carried out enzyme immunoassay to measure VP in the serum.
By measuring the amount of F and finding that the transition from cirrhosis to liver cancer can be monitored, the present invention was completed. That is, the present invention relates to a method for testing liver cancer, which comprises measuring the amount of vascular endothelial cell growth factor / vascular permeability factor in human serum, and specifically reacts with vascular endothelial cell growth factor / vascular permeability factor. The present invention relates to a liver cancer test agent characterized by comprising: The method for testing liver cancer of the present invention is particularly suitable for monitoring the transition from cirrhosis to liver cancer.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. A well-known labeled immunoassay using a substance that specifically reacts with VPF, for example, an anti-VPF antibody or a VPF receptor, is suitable for measuring the VPF concentration in serum, and is also a preferable method in the present invention. For example, when an anti-VPF antibody is used, erythrocytes, latex, radioisotopes, enzymes, luminescent substances, fluorescent substances, metal molecules, metal gels,
Labeled immunoassay using bacteriophage or the like is applied. In particular, in clinical applications, enzyme immunoassays are preferred, and in practice, enzyme immunoassays are widely used. Enzyme-linked immunosorbent assay (EIA) is a method of tracking an antigen-antibody reaction using an enzyme activity as an index, and measuring the amount of an antigen or an antibody therefrom.For details, see, for example, Kitagawa et al. 31 Protein Nucleic Acid Enzyme Separate Volume 1987. This measurement method
Competitive, non-competitive, homogeneous, and heterogeneous methods are classified according to differences in the measurement target, labeling substance, type of antigen-antibody reaction, and method of separating conjugate / free form (B / F separation method). Have been.

[0010]

【Example】

(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 resulting protein was used as an antigen to prepare rabbit anti-VPF according to a conventional method. Polyclonal antibodies were made.
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 ligated to peroxidase (horseradish) by the hinge method and labeled with peroxidase. A rabbit anti-VPF polyclonal antibody was obtained.

(3) Serum VP of cirrhosis and liver cancer patients
Measurement of F concentration The serum VPF concentration of 40 cirrhosis patients and 50 liver cancer patients was measured by an enzyme immunoassay as shown below. That is, an anti-VPF polyclonal antibody (5 μg
/ ml) in a 96-well plate at 100 μl / well and left overnight at 4 ° C., followed by 0.1% bovine serum albumin (BS
A), washed 4 times with PBS. After blocking with 1% BSA, 0.1 M sodium chloride, 0.1% sodium azide, and 0.1 M sodium carbonate buffer (pH = 6.5) 1 (37 ° C. for 4 hours), 1% BSA, 0.1 M 3% with 50 mM sodium phosphate buffer (pH = 7.0) (sample diluent) containing 0.4% gelatin, 1 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1 M sodium chloride and 0.1% sodium azide. Serum diluted 1: 2 or standard VPF dissolved in the same sample diluent was added and left at room temperature for 1 hour.
After washing 6 times with 0.1% BSA and PBS, 100 μl / well of peroxidase-labeled anti-VPF polyclonal antibody was added and reacted at room temperature for 1 hour. Again, 0.1% BS
A. After washing 8 times with PBS, 0.125% (w / v) orthophenylenediamine, 0.015% hydrogen peroxide, 0.2 M tris (hydroxymethyl (aminomethane) -citrate buffer (p
H = 5.2) was added at 100 μl / well, and reacted at room temperature for 30 minutes. After adding 2N sulfuric acid at 100 μl / well to stop the reaction, the absorbance at 490 nm relative to the absorbance at 650 nm was measured using a plate reader (M-Vmax, Molecular Device).
s company).

Table 1 shows the results of measurement of serum VPF levels in 40 patients with cirrhosis and 50 patients with liver cancer. V of cirrhosis patients
The PF amount (mean ± standard deviation) was 41.4 ± 38.0 pg / ml, whereas that of liver cancer patients was 70.4 ± 52.2 pg / ml, which was significantly higher in liver cancer patients than in liver cirrhosis patients. Was. From this, it became clear that liver cancer can be tested by measuring the amount of VPF in serum.

[0014]

[Table 1]

[0015]

According to the present invention, that is, by measuring the amount of VPF in serum using a labeled immunoassay,
Liver cancer can be tested, and the transition from cirrhosis to liver cancer can be monitored (diagnosed). The present invention can be used as a new test method in addition to the test methods used so far.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Katsuhiko Matsuo 2nd Okubo Tsukuba, Ibaraki Pref.

Claims (3)

[Claims] 1. A method for testing liver cancer, comprising measuring the amount of vascular endothelial cell growth factor / vascular permeability factor in human serum. 2. A liver cancer test agent comprising a substance that specifically reacts with vascular endothelial cell growth factor / vascular permeability factor. 3. The substance which specifically reacts with vascular endothelial cell growth factor / vascular permeability factor is an anti-vascular endothelial cell growth factor / vascular permeability factor antibody or a vascular endothelial cell growth factor / vascular permeability factor receptor. The test agent for liver cancer according to claim 2, which is present.