JPH08313522A - Method for measuring vascular permeability factor - Google Patents

Abstract

PURPOSE: To accurately measure the amount of VPF(vascular permeability factor) by using the chemiluminescence as a detection method of enzyme immunity measurement using anti-VPF polyclonal antibody. CONSTITUTION: Anti-VPF polyclonal antibody (5μg/ml) is poured into each hole of 96-hole plate, is left stand for one night at approximately 4 deg.C, is washed in phosphoric acid buffering physiological salt solution containing 0.1% bovine serum albumin(BSA), and is blocked for approximately one hour in 1% BSA- containing buffer solution. The serum of a healthy person which is diluted approximately ten times by sodium phosphate buffer solution containing 1% BSA is added and is left stand for approximately one hour. It is washed with tris buffer solution, alkali phosphatase labeled antibody VPF polyclonal antibody is added to react for approximately one hour, and is washed with tris buffer liquid. For example, Lumi-Phos 530 which is diluted by approximately five times in a buffer solution is added into it, and the entire part is left stand for approximately 30 minutes at approximately 37 deg.C. After that, by measuring luminous intensity, a small amount of VPF in the humor derived from a human can be measured.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention is present in body fluids such as blood and urine, and is known as a factor for inducing angiogenesis. It is a vascular endothelial growth factor or vascular permeability factor (vas
cular endothelial growth factor or vascular permea
The present invention relates to a method for measuring the amount of a factor called a bility factor (hereinafter abbreviated as VPF) and can be an effective means in diagnosis or pathology.

[0002]

2. Description of the Related Art When a physiologically active substance existing in a living body, that is, a trace amount of a substance showing a pharmacological action in a very small amount, is used as a diagnostic marker or an index for determining a therapeutic effect or monitoring after operation, it is highly sensitive. A different measurement system is required. As one of such measurement systems, a radioimmunoassay (RIA) using a radioisotope has been used for a long time. Although this method has high sensitivity and high specificity and is easy to perform an analytical operation, it is limited in handling due to the use of radioactive substances, the equipment and facilities are expensive, and the disposal after use is troublesome. Therefore, recently, a non-radioactive immunoassay using a non-radioactive substance as a label has been developed. In particular, enzyme immunoassays that utilize the amplifying action of enzymes are widely used because of their high sensitivity. This method is generally called an enzyme immunoassay, but the detection method used in this method can be roughly classified into a colorimetric method, a fluorescent method, and a chemiluminescent method. Comparing the detection sensitivities of these methods, it is said that the colorimetric method, the fluorescence method, and the chemiluminescence method have higher sensitivity in this order, and as a detection method in the enzyme immunoassay,
It is generally said that the chemiluminescence detection method has better detectability, that is, the ability to measure a smaller amount of substance, than the colorimetric detection method, but when constructing an enzyme immunoassay method, Effective detection methods differ depending on the amount of antigen contained in the sample, the affinity of the antibody used, and the measurement system.Therefore, there are various measurement methods depending on the substances to be measured. The current situation is that it must be done.

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. Known to
[(Folkman, J., Cancer Res. 46: 467 (1986)]], a basic fibroblast growth factor that directly acts on vascular endothelial cells as a factor that induces angiogenesis.
actor, bFGF), acidic fibroblast growth factor
ast growth factor, aFGF), platelet-derived endothelial cell growth facto
r, PD-ECGF), etc., and transforming growth factor-α (TGF-
α), transforming growth factor-β (TGF-β), angiog
enin, tumor necrosis factor-α (TNF-α), etc. have been found [Folkman, J. & Shing, 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 it is clear that it is present in brain, pituitary gland, kidney and ovary in different tissues. Has been [[Ferrar
a, N., et.al. Endocrine Reviews 13:18 (1992)], Human VPF is angiogenesis and metastasis of breast cancer [Weider, N, et.al. N.En.
gl.J.Med. 324: 1 (1991)] and angiogenesis of renal cell carcinoma [Medical progress, 168: 231 (1994)], or angiogenesis in retinal diseases [Adamis, AP et.al., Biochem. Biophys.Res.Com
m., 193: 631 (1993)]. From these facts, measuring the amount of VPF in human-derived body fluids such as serum, plasma, urine, saliva, bone marrow fluid, ascites fluid or tissue extract is useful for diagnosing cancer and predicting metastasis or determining therapeutic effect. May be useful to.

The cDNA for the human VPF gene
Has been isolated, the base sequence has been determined, and the 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 types) were produced, and among them, those with 121 amino acid residues (VPF121) and 1
It is said that a protein with 65 amino acid residues (VPF165) is a mature protein [(Ferrara, N., et.al. Endocrine
Reviews 13:18 (1992)]. VPF121 has a deletion of 44 amino acids at the carboxyl terminus of VPF165. It is not clear whether VPF121 and VPF165 have different actions on vascular endothelial cells, but a monoclonal antibody against human VPF121 has already been identified. Obtained by the present inventors, it has been clarified that VPF can be measured by an enzyme immunoassay using the monoclonal antibody and a polyclonal antibody against human VPF121 (Japanese patent: peptide and monoclonal antibody, application date Heisei June 10, 6). However, with this assay, V in human body fluids such as serum
It is difficult to measure the amount of PF accurately, the measurement method suitable for VPF measurement is unknown, and there is a current demand for a method that can be measured accurately.

[0006]

Therefore, the present inventors
When measuring the amount of VPF in a human-derived body fluid, it was examined which detection method, colorimetric detection method or chemiluminescence detection method, and under what conditions are appropriate.

[0007]

[Means for Solving the Problems] The present inventors constructed an enzyme-linked immunosorbent assay for measuring VPF using an anti-VPF polyclonal antibody using a serum sample of a healthy person and a urine sample of a healthy person as samples, and used it as a detection method. The inventors have found that the amount of VPF can be accurately measured by using the chemiluminescence method, and have completed the present invention.

That is, the present invention relates to a method for measuring VPF, which uses chemiluminescence for detection in the enzyme immunoassay method.

The method for measuring VPF of the present invention is a chemiluminescent enzyme immunoassay using an antibody against VPF, and the enzyme immunoassay is a sandwich method using a monoclonal antibody or a polyclonal antibody against human VPF. Can be applied. The chemiluminescence measurement is a method of measuring light generated by using a luminescent substrate as a substrate of an enzyme.

[0010]

EFFECTS OF THE INVENTION According to the present invention, VPF in human-derived body fluid such as serum, plasma, urine, saliva, bone marrow fluid, ascites fluid or tissue extract can be measured with high sensitivity even with a trace amount that could not be measured until now. can do.

[0011]

The present invention will be described in detail based on the following examples. (1) Preparation of Anti-VPF Polyclonal Antibody The isolated human VPF cDNA was produced in E. 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 A VPF polyclonal antibody was prepared. Rabbit serum with increased antibody titer was separated and an IgG fraction of 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 by the hinge method and peroxidase-labeled rabbit anti-VPF polyclonal. An antibody was obtained. Similarly, a portion of the IgG fraction was digested with pepsin to prepare F (ab ') 2, which was then linked with alkaline phosphatase (derived from bovine small intestine) by the hinge method to give a rabbit anti-VPF polyclonal antibody labeled with alkaline phosphatase. Obtained.

(3) Measurement of detectability of enzyme immunoassay using colorimetric detection VP in serum using rabbit anti-VPF polyclonal antibody
The method for measuring the F content was constructed as follows. Ie 5
1 μg / mL anti-VPF polyclonal antibody (dissolved in 25 mM sodium carbonate buffer pH 9.0 containing 0.1 M sodium chloride) was applied to a 96-well enzyme immunoassay plate.
Add 00 μL each and let stand overnight at 4 ° C for anti-VP
The F polyclonal antibody was adsorbed on the plate. 0.1%
Phosphate buffered saline containing bovine serum albumin
After washing the wells of the plate 6 times with (0.1% BSA, PBS), phosphate-buffered saline containing 1% bovine serum albumin (1% BSA, PBS) was placed in the wells at room temperature for 1 hour. Left for hours. After removing 1% BSA and PBS from the hole, 1
% Bovine serum albumin, 0.4% gelatin, 1 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate,
Serum diluted 10 times with 50 mM sodium phosphate buffer pH 7.0 (specimen diluent) containing 0.1 M sodium chloride and 0.1% sodium azide, or standard VPF dissolved in the same specimen diluent was added at room temperature. It was left for 1 hour. 0.1% BS
After washing 6 times with A and PBS, 100 μL of peroxidase-labeled rabbit anti-VPF polyclonal antibody dissolved in 1% BSA and PBS was added to each well and left at room temperature for 1 hour. 0.1%
After washing 6 times with BSA and PBS, 0.15 M containing 0.2 mg / mL orthophenylenediamine and 0.015% hydrogen peroxide
Color was developed by adding 100 μL of citrate buffer (pH 5.0). The reaction was stopped by adding 50 μL of 2N sulfuric acid, and then the absorbance (OD490 / 650) was measured. Human VPF expressed in yeast was used as a standard substance (Japanese patent: method for producing vascular permeability factor, application date July 21, 1993,
See application number 05-200181). The results of measurement using the standard substance were plotted on a graph to obtain FIG. The value of [the average value of the measured value of the sample diluent + 2 times the standard deviation] and the value of [the average value of the measured value of the standard VPF-the standard deviation 2 times] of each concentration
Was calculated, and the minimum value of the standard VPF values showing a difference between the two values was taken as the detectability (detection limit value), the detectability of the colorimetric detection method was 50 pg / ml. In addition, the amount of VPF in the serum of healthy subjects was measured by this method, but most of the samples were below the detectability (experimental results not shown).

(4) Measurement of detectability of enzyme immunoassay using chemiluminescence detection method Anti-VPF polyclonal antibody (5 μg / mL) was added at 100 μL /
Spread well on a 96-well plate and leave at 4 ° C overnight,
The plate was washed 4 times with 0.1% BSA and PBS. 1% BSA, 0.
Blocking with 1M sodium chloride, 0.1% sodium azide, 0.2M sodium carbonate buffer pH 9.5 (1 at room temperature
Time), 50 mM sodium phosphate buffer containing 1% bovine serum albumin, 0.4% gelatin, 1 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1M sodium chloride, 0.1% sodium azide.
Serum of a healthy person diluted 10 times with 7.0 (specimen diluent) or standard VPF dissolved in the same specimen diluent was added and left at room temperature for 1 hour. Tris buffer containing 0.05% Tween 20, 0.14 M sodium chloride, 5 mM potassium chloride, pH 7.4
After washing 4 times with (TBS-T), 100 μL / well of alkaline phosphatase-labeled anti-VPF polyclonal antibody was added and reacted at room temperature for 1 hour. After washing 4 times with TBS-T and twice with Tris buffer pH 7.4 containing 0.14 M sodium chloride and 5 mM potassium chloride, 0.1 M diethanolamine buffer containing 1 mM magnesium chloride and 0.02% sodium azide. 100 μl / w of Lumifos 530 (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 5 times with liquid pH 10.0
Each ell was placed and left at 37 ° C. for 30 minutes, and then the luminescence intensity was measured. The result of measurement using the standard substance was plotted in a graph to obtain FIG. Calculate the value of [average value of sample diluted solution + 2 times standard deviation] and the value of [average value of standard VPF measured value – 2 times standard deviation] for each concentration, and see the difference between them. If the minimum value of the standard VPF values to be detected is the detectability,
The detectability of the chemiluminescent detection method was 1.0 pg / ml, which was 50 times higher than that of the colorimetric detection method.

(5) Measurement of VPF amount in serum of healthy subjects by enzyme immunoassay using chemiluminescence detection 30 serum samples of healthy subjects (23 males, 7 females) by enzyme immunoassay using chemiluminescent detection The amount of VPF in human) is measured,
The results are plotted in a graph as shown in FIG. The amount of VPF in serum of healthy subjects was in the range of 8.1 to 35.8 pg / ml, and the average value was about 19 pg / ml. From the above,
By using the chemiluminescence detection method, it became possible to measure the amount of VPF in serum which could not be measured by the colorimetric detection method.

(6) Measurement of VPF content in urine samples of normal subjects by enzyme immunoassay using chemiluminescence detection method Using chemiluminescence detection method using 114 urine samples of healthy subjects (29 men, 85 women) The amount of VPF in urine was measured by the enzyme immunoassay. First, anti-VPF polyclonal antibody (5
100 μl / well of each 100 μl / well was spread on a 96-well plate and left overnight at 4 ° C., and then washed 4 times with 0.1% BSA and PBS. 1% BSA, 0.1M sodium chloride, 0.1% sodium azide, 0.2M sodium carbonate buffer pH 9.5
After blocking (1 hour at room temperature) with 1% bovine serum albumin, 0.4% gelatin, 1 mM magnesium chloride, 2
50mM sodium phosphate buffer containing 0mM sodium ethylenediaminetetraacetate, 0.1M sodium chloride and 0.1% sodium azide pH 7.0 (specimen diluent) I left it. After washing 4 times with TBS-T, 100 μl / well of alkaline phosphatase-labeled anti-VPF polyclonal antibody was added to each well at room temperature for 1 hour.
Allowed to react for hours. Tris buffer containing 0.14 M sodium chloride and 5 mM potassium chloride 4 times with TBS-T.
After washing twice with H7.4, 1 mM magnesium chloride, 0.0
Lumiphos 530 diluted 5 times with 0.1 M diethanolamine buffer pH 10.0 containing 2% sodium azide
(Manufactured by Wako Pure Chemical Industries, Ltd.) was put in 100 μl / well each, left at 37 ° C. for 30 minutes, and then the emission intensity was measured. The results are plotted in a graph as shown in FIG.
The amount of urinary VPF was measured by separately measuring the creatinine concentration in urine in order to correct the concentration of urine, and was shown as the concentration per 100 (mg / dl) creatinine (pg / ml). Urinary VPF of healthy subjects
The amount is in the range of 0-91.6 (pg / ml) / 100 (mg / dl) creatinine, and the average value is about 25 (pg / ml) / 100 (mg / d
l) It was creatinine. As described above, the amount of VPF in urine was successfully measured by the chemiluminescence detection method.

[0017]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to measure a very small amount of VPF in human-derived body fluids such as serum, plasma, urine, saliva, bone marrow fluid, ascites fluid or tissue extract, and to prevent tumors, inflammation, etc. Since VPF can be used for determination of diagnostic markers and therapeutic effects, or for post-surgical monitoring, it has an excellent effect of being widely used as a diagnostic agent or for pathological applications. It is a thing.

[Brief description of drawings]

FIG. 1 is a diagram showing the detection ability of VPF using a standard VPF by an enzyme immunoassay method using a colorimetric detection method.

FIG. 2 is a diagram showing the detection ability of VPF using a standard VPF by an enzyme immunoassay using chemiluminescence detection.

FIG. 3 is a diagram showing a histogram of the results of measuring the amount of VPF in serum of healthy subjects by an enzyme immunoassay method using chemiluminescence detection method.

FIG. 4 is a diagram showing the results of measuring the amount of VPF in urine of healthy subjects by an enzyme immunoassay using chemiluminescence detection.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shinichi Kondo No.2 Okubo, Tsukuba-shi, Ibaraki Toagosei Co., Ltd. (72) Inventor Iwao Omori No.2 Okubo, Tsukuba-shi, Ibaraki Toagosei Co., Ltd.

Claims (1)

[Claims] 1. A method for measuring a vascular permeability factor, which comprises using chemiluminescence for detection in an enzyme immunoassay.