JPH0933531A - Method for deciding presence of lung cancer cell and examination medicine for diagnosing lung cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a marker for diagnosing serum useful for early diagnosis of lung cancer and judgment of the effect of treatment by measuring the quantity of vascular penetration factors(VPF) existing in a human serum. SOLUTION: Preferably, a labeled immunoassay where the antigen antibody reaction using a polyclonal or monoclonal antibody for VPF is applied, especially a chemiluminescence detection system enzyme immunoassay is employed for measuring the concentration of VPF in a serum. The immunoassay employs a luminescent enzyme substrate and measures the luminescence thus determining the concentration of VPF in a serum with high sensitivity. Since the VPF to be measured is essential for proliferation and development of lung cancer cells, a serum tumor marker insusceptible to the type of tissue and highly useful for early diagnosis can be obtained. It is also effective as a factor for judging the effect of treatment because it has tight relationship with proliferation and development of lung cancer cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vascular permeability factor or a vascular endothelial growth factor present in serum.
The present invention relates to a method for determining whether or not the serum donor has lung cancer cells based on the measurement of the abundance of a factor called an actor) (hereinafter abbreviated as VPF), in which a doctor diagnoses lung cancer. The present invention relates to a method for providing useful information when carrying out the method, a test agent for diagnosing lung cancer useful for diagnosing lung cancer, and is used in the pharmaceutical industry.

[0002]

2. Description of the Related Art At present, X-ray examination is generally used as an early diagnosis of lung cancer. On the other hand, studies on serum tumor markers have been conducted, and CEA (carcinoembryonic
antigen), SCC (squamous cell carcinoma-related a
ntigen), NSE (neurospecific enolase), SLX (si
Alyl LeX-i antigens) have been reported, but their positive rate in clinical stage I is about 30%, even though they are high (Hiroshi Ariyoshi, Masayoshi Kuwahara: clinical application of tumor markers,
Lung cancer, pathology and clinic, 8, Extra edition 289-299, 1990).
Therefore, these markers are not suitable for early diagnosis. This can be caused by
Many of these tumor markers have different positive rates depending on the tissue type, that is, SCC is squamous cell carcinoma, SLX
Shows high positive rate in adenocarcinoma and NSE in small cell carcinoma, but low positive rate in other tissue types. Therefore, the expression levels of these tumor markers are different in cells of different tissue types. It is considered that. On the other hand, angiogenesis, the proliferation of capillary endothelial cells,
It is known that the phenomena of migration and tissue infiltration play important roles in physiological and pathological phenomena such as fetal growth, wound healing, and cancer cell proliferation [(Fo
lkman, J., Cancer Res. 46: 467 (1986)], as a factor that induces angiogenesis, basic fibroblast growth factor (basic fibroblast growth factor) is a substance that directly acts on vascular endothelial cells.
growth factor (bFGF), acidic fibroblast growth factor (acidic
fibroblast growth factor, aFGF), vascular endothelial growth factor
actor / vascular permeability factor (VPF), platelet-derived endothelial cell growth factor
cell growth factor (PD-ECGF), etc., is also a substance that indirectly acts on vascular endothelial cells.
factor-α (TGF-α), transforming growth factor-β (T
GF-β), angiogenin, tumor necrosis factor-α (TNF-
α) etc. have been found [Folkman, J. & Shing, Y., J.
Biol. Chem., 267: 10931 (1992)].

Of 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, 168: 231 (1994)] or retinal disease [Adamis, AP et.al., Biochem.B
iophys.Res.Comm., 193: 631 (1993)]. C for human VPF gene
DNA 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 from this gene (121 amino acid residues,
165, 189, and 206 types) were created, and among them, those with 121 amino acid residues (VPF12
1) and 165 amino acid residues (VPF165) are said to be mature proteins [(Ferrara, N., et.al. End
ocrine Reviews 13:18 (1992)]. VPF121 is VPF165
It has a deletion of the 44 amino acids at the carboxyl terminus, but it is not clear whether VPF121 and VPF165 have different effects on vascular endothelial cells. This monoclonal antibody against human VPF121 has already been obtained by the present inventors, 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: Peptide and Monoclonal antibody, filing date June 10, 1994).
In addition, the present inventors have already established a method for detecting VPF in the enzyme immunoassay, and it has been clarified that VPF of several pg / ml can be detected (Japanese Patent Application No. Hei 7).
-141271).

[0004]

Based on the above findings, the present inventors have conducted earnest studies to find a tumor marker useful as a tumor marker, particularly for the diagnosis of early lung cancer. .

[0005]

As a result of various investigations, the present inventors have found that the above VPF is useful as a tumor marker, particularly as a tumor marker useful for diagnosing early lung cancer. The invention was completed. That is, the present invention is an invention relating to a method for determining the presence or absence of lung cancer cells, which is based on the amount of vascular permeability factor present in human serum, and a lung cancer diagnosis characterized by comprising an antibody of vascular permeability factor. The invention relates to an inspection drug for medical use.

[0006]

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 detection system enzyme immunoassay that the present inventors have previously applied for a patent, which makes it possible to measure VPF in serum with high sensitivity, which is useful for diagnosing lung cancer. The 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.

[0007]

According to the clarification by the present inventors, V
Unlike conventional lung cancer markers in serum, PF is essential for the growth and progression of cancer cells, so it is considered that PF is expressed in all cancer cells regardless of tissue type. Unlike that, it is highly useful as a serum tumor marker for the purpose of early diagnosis without being affected by tissue type, and further, because it is closely related to the growth and progression of cancer cells, It exhibits an action as an excellent serum tumor marker that can be used as a therapeutic effect determination, follow-up observation or prognostic factor.

[0008]

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

(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 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 lung cancer patient The VPF concentration in serum of lung cancer patient was measured by enzyme immunoassay using chemiluminescence detection method as shown below. 100 μl / w of anti-VPF polyclonal antibody (5 μg / ml)
Spread each ell on a 96-well plate and leave at 4 ° C overnight,
The plate was washed 4 times with 0.1% bovine serum albumin (BSA), PBS. 1% BSA, 0.1M sodium chloride, 0.1%
Sodium azide, 0.2M sodium carbonate buffer pH
After blocking with 9.5 (1 hour at room temperature), 1% bovine serum albumin, 0.4% gelatin, 1 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1%.
50mM sodium phosphate buffer containing 1M sodium chloride and 0.1% sodium azide pH 7.0 (specimen diluent)
Serum (lung cancer patient or healthy person) diluted 10-fold with or standard VPF dissolved in the same specimen diluent was added and left at room temperature for 1 hour. Tris buffer pH 0.05% Tween 20, 0.14M sodium chloride, 5 mM potassium chloride
After washing 4 times with 7.4 (TBS-T), 100 μl / w of alkaline phosphatase-labeled anti-VPF polyclonal antibody was used.
Each ell was added and reacted at room temperature for 1 hour. 4 with TBS-T
After washing twice with Tris buffer pH 7.4 containing 0.14M sodium chloride and 5 mM potassium chloride, 1m
Lumiphos 530 (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 5 times with 0.1 M diethanolamine buffer containing 10.0 M magnesium chloride and 0.02% sodium azide was used.
After adding each 00 μl / well and leaving at 37 ° C. for 30 minutes, the emission intensity was measured. The measurement results of the serum VPF concentration of 28 lung cancer patients are shown in FIG. 1 as a scatter diagram. The high value sample was about 300 pg / ml, and the average value was 84.7 pg / ml. On the other hand, as a result of measuring the VPF concentration in serum of 290 healthy subjects in the same manner, it was about 120 pg / ml in the high value sample,
The average value was 24.9 pg / ml (Fig. 2). From the above, it can be said that lung cancer patients have significantly higher VPF concentration in serum than healthy subjects. In addition, VPF in serum of healthy subjects
The positive rate in lung cancer patients was calculated to be 61% using the cutoff value as the average value of the concentration plus twice the standard deviation value, which can be used as a serum diagnostic marker.

[0011]

INDUSTRIAL APPLICABILITY The VPF, which was found by the present inventors and is excellent as a lung cancer marker in serum, can be determined by measuring it, particularly by using a chemiluminescence-detecting enzyme immunoassay with high sensitivity. It is very useful for the early diagnosis of, the determination of the therapeutic effect, and the follow-up observation.

[Brief description of drawings]

FIG. 1 is a scatter diagram of the measurement results of the amount of VPF in serum of 28 serum samples of lung cancer patients by an enzyme immunoassay using a chemiluminescence detection system.

FIG. 2 is a scatter diagram of the measurement results of the amount of VPF in serum of 290 healthy human serum samples by an enzyme immunoassay using a chemiluminescence detection system.

Claims (2)

[Claims] 1. A method for determining the presence or absence of lung cancer cells, which is based on the amount of vascular permeability factor present in human serum. 2. A diagnostic agent for diagnosing lung cancer, which comprises an antibody of a vascular permeability factor.