JPH1048218A - Method and reagent for inspecting effect of treatment of liver cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To monitor the effect of treatment after application of liver artery embolus 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. The immunoassay is classified to competitive immunoassay, noncompetitive immunoassay, homogenious immunoassay, heterogenious immunoassay, etc., depending on the object to be assayed, the labeling substance, the type of antigen-antibody reaction, the separating method of bonded body/free body.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the use of vascular endothelial grow factor in blood serum.
th factor / vascular permeability factor, below VEG
The present invention relates to a method for testing the therapeutic effect of liver cancer by measuring the amount of F / VPF or VPF) and a test agent used therefor. The therapeutic effect test for liver cancer according to the present invention is performed by performing hepatic artery embolization (TAE). It is used for monitoring the therapeutic effect later, and relates to medical diagnostic technology and test drug technology.

[0002]

2. Description of the Related Art Unlike other solid tumors, noninvasive treatments such as percutaneous ethanol injection therapy (PEIT) and hepatic artery embolization (TAE) play an important role in treating liver cancer. Play. This is because there are few cases that can be resected due to multiple lesions and complicated cirrhosis (Shuichiro Shiina et al., Medical History, Vol.171, No.14, p.1139-1144 1994
Year). As a method of monitoring the effect after hepatic artery embolization (TAE) is performed, it is usually performed by an X-ray CT examination, a histopathological examination, or the like, and none is used as a serodiagnosis.

In addition, angiogenesis, ie, 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 normal or tumor cell lines of mouse, rat, guinea pig, bovine and human, 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. J. Me.
d. 324: 1 (1991)] and angiogenesis of renal cell carcinoma [Ayumi of Medicine 1
68: 231 (1994)], or angiogenesis in retinal disease [Ad
amis AP et. al., Biochem. Biophys. Res. Comm. 1
93: 631 (1993)].

On the other hand, in malignant tumors, it has been clarified that VPF is localized in the ischemic region of the tissue, which suggests that local hypoxia induces the expression of VPF. (Shweiki D. et.al. Nature (L
ondon) 359: 843-5 (1988)). It has also been reported that hypoxia is an important angiogenic stimulator in glioma, breast cancer, coronary artery disease, and diabetes (Shwei
ki D. et.al.Nature (London) 359: 843-5 (1988), Hla
tky L. et.al.Cancer Res. 54: 6083-6086 (1994), Sab
ri M. et.al. Am. Heart.J. 121: 876-880 (1991), Aie
llo LPN Engl. J. Med. 331: 1480-1487 (1994)).

Further, regarding the human VPF gene,
DNA has already been isolated, its nucleotide sequence has been determined, and its amino acid sequence has been deduced. In this gene, four kinds of proteins with different numbers of amino acid residues (four kinds of amino acid residues 121, 165, 189, and 206) are produced from one gene, and 121 amino acids are included in them. Residue number (VPF ₁₂₁ ) and 165 amino acid residue number
(VPF ₁₆₅ ) is said to be a secreted protein [(Ferrara
N. et. Al. Endocrine Reviews 13:18 (1992)]. VPF
₁₂₁ lacks the 44 amino acids at the carboxyl terminus of VPF ₁₆₅ , but it is not clear whether VPF ₁₂₁ and VPF ₁₆₅ have different effects on vascular endothelial cells.

On the other hand, a monoclonal antibody against human VPF ₁₂₁ has already been obtained by the present inventors (Japanese Patent Application No. 6-152805, "Peptides and Monoclonal Antibodies.")
By an enzyme immunoassay using a polyclonal antibody against F _121, VPF number pg / ml is also apparent is can be measured (Japanese Patent Application No. 7-141271, "method of measuring vascular permeability factor".

[0008]

Under the circumstances described above, the present inventors measured the amount of VPF in the serum of a liver cancer patient to obtain a therapeutic effect on liver cancer, particularly hepatic artery embolization.
The purpose of this study was to examine whether it was possible to monitor the effect of treatment after (TAE). That is, an object of the present invention is to provide a method for monitoring the therapeutic effect of liver cancer, in particular, the therapeutic effect after hepatic artery embolization (TAE), which is a method of treating liver cancer.

[0009]

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.
The amount of F was measured and the amount of serum VPF and hepatic artery embolization (TA
E) The inventors have found that there is a strong correlation between the therapeutic effects after the operation and completed the present invention. That is, the present invention relates to a method for testing the therapeutic effect of liver cancer, which comprises measuring the amount of vascular endothelial cell growth factor / vascular permeability factor in human serum, and a method specifically for vascular endothelial cell growth factor / vascular permeability factor. The present invention relates to a therapeutic effect test agent for liver cancer, which is characterized by comprising a substance that reacts with liver cancer. In particular, the present invention relates to a method for testing the therapeutic effect of liver cancer, which is suitable for a method for monitoring the effect after hepatic artery embolization (TAE).

[0010]

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.

[0011]

【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 bound to peroxidase (horseradish) by the hinge method and labeled with peroxidase. A rabbit anti-VPF polyclonal antibody was obtained.

(3) Changes in serum VPF concentration during hepatic artery embolization (TAE) Serum VPF after hepatic artery embolization (TAE) in 6 patients with liver cancer
Changes in PF concentration were examined by enzyme immunoassay as described below. That is, an anti-VPF 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 0.1% bovine serum albumin (B
SA) and washed 4 times with PBS. After blocking (4 hours at 37 ° C.) with 1% BSA, 0.1 M sodium chloride, 0.1% sodium azide, and 0.1 M sodium carbonate buffer (pH = 6.5), 1% BSA, 0.1 M sodium chloride was added. 3 times with 50 mM sodium phosphate buffer (pH = 7.0) (sample diluent) containing 4% gelatin, 1 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1 M sodium chloride and 0.1% sodium azide. The diluted serum or the 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 Devices).
(Manufactured by the company).

[0014] Hepatic artery embolization (T
AE) After treatment, the results of daily measurement of the amount of serum VPF are shown in FIG. After 7 days, the serum VPF level increased about 5-fold as compared to before the TAE treatment (0 day).
From this, TPF can be determined by measuring the amount of serum VPF.
It has been clearly shown that the therapeutic effect of AE therapy can be monitored.

[0015]

According to the present invention, that is, the effect of hepatic artery embolization (TAE) treatment can be monitored by measuring the amount of serum VPF using a labeled immunoassay. The test can be used as an index for effectively treating hepatic artery embolization (TAE). Further, the present invention is very useful because it can be performed more simply and efficiently than the X-ray CT inspection method used so far. Further, in addition to the conventional method, it can be used as a new inspection method.

[Brief description of the drawings]

FIG. 1 shows the results of daily measurement of serum VPF levels by enzyme immunoassay after hepatic artery embolization (TAE) treatment in six hepatic cancer patients treated with hepatic artery embolization (TAE). FIG.

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

Claims (3)

[Claims] 1. A method for testing the therapeutic effect of liver cancer, comprising measuring the amount of vascular endothelial cell growth factor / vascular permeability factor in human serum. 2. A therapeutic effect test agent for liver cancer, which comprises 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. 3. The therapeutic effect test agent for liver cancer according to claim 2, wherein: