JP2868841B2 - Method and kit for detecting GMP abnormality - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to GMP represented by human thrombocytopenia, human diabetes, human kidney disease, human arteriosclerosis, and human thrombosis.
The present invention relates to a method for detecting abnormal production of -130 and a kit for quantification thereof.

[Conventional technology]

In order for blood flow to maintain fluidity, the action of vascular endothelial cells and platelets plays an important role. When the vascular endothelium is damaged in the human body and the endothelial tissue is exposed, platelets in the blood adhere to this site and release various platelet stimulating substances. These substances activated new platelets. It is believed that thrombus is formed by agglutinating on previously adhered platelets. The formation of thrombus is considered to be a cause of the onset of myocardial infarction and cerebral thrombosis and the development of arteriosclerosis. Thus, the detection of activation or damage of vascular endothelial cells and platelets has clinical significance.

Recently, GMP-140 present in the Weibel-Palade body of the α-granules of platelets and vascular endothelial cells.
(Stenberg PE et al., Journal of Cell Biology (J. Cell. Biol.), No. 1) was found to appear on the membrane surface with the activation of platelets and vascular endothelial cells. 101, 880 (1985)]. Furthermore, it is described that the number of activated platelets and the number of thrombus sites can be measured by a flow cytometry method or an in vivo (in vivo) imaging method using an antibody specific to GMP140 [Johnston (Johnston)
ston) GI et al., Blood, Vol. 69, p. 1401 (1987), Palabrica TM et al., Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sc)
i.USA), 86, 1036 (1989)]. On the other hand, GMP-14
0 DNA and amino acid sequences were also reported, indicating that not only membrane-bound GMP-140 but also unbound or free GM
The possible presence of P-140 has been suggested [Johnston GI et al., Cell, 56, 1033 (1989)].

[Problems to be solved by the invention]

However, these reports merely describe changes in GMP-140 on the membranes of platelets and vascular endothelial cells. In addition, the association between human disease and changes in GMP-140 on platelet membranes is related to adult respiratory distress syndrome (adult respiratory syndrome).
Only distress syndrome has been reported [George JN et al., Journal of Clinical Investigation (J. Clin. Inves).
t.], Vol. 78, p. 340 (1986)], and other reports
The results are in vitro or in animal experiments.

Furthermore, the existence of free GMP-140 was only a hypothesis. To date, there have been no reports demonstrating the presence of free GMP-140, and no further reports on the relationship between free GMP-140 production and a specific disease.

In addition, a competitive radioimmunoassay method [Berman CI et al.,
Methods in Enzym
169, 321 (1989)] and a sandwich enzyme immunoassay method using two types of monoclonal antibodies (JP-A-3-150465). It describes only the method for measuring the membrane-bound GMP-140 extracted and purified, but does not describe the reactivity with free GMP-140 or the relationship with disease.

An object of the present invention is to provide a novel measurement method for detecting a disease associated with abnormal production of free GMP-140 using free GMP-140 as an index, and a kit for quantification.

[Means for solving the problem]

In summary, the first invention of the present invention is a GMP-130
Is characterized by having the following physicochemical properties.

(B) The molecular weight analyzed by the SDS-PAGE method is 130 kDa.

(B) A 40 kDa fragment was obtained by trypsin digestion, and the N-terminal amino acid sequence was AFQHQS.

(C) fragments of 60kDa was obtained by V ₈ protease degradation, the N-terminal amino acid sequence is, HXLKKKXA
L.

Further, the second invention of the present invention relates to a method for detecting a disease associated with abnormal production of GMP-130, wherein the amount of GMP-130 having the physicochemical properties (a) to (c) of the first invention in plasma is determined. It is characterized by comparison with a normal value.

Further, the third invention of the present invention provides the GMP of the first invention.
-130 For a kit for detecting a disease associated with abnormal production, anti-GMP
-130 antibody.

In view of the above situation, the present inventors have conducted intensive studies and found that free GMP-140-like substance,
The presence of P-130 was discovered. Next, by measuring the amount of GMP-130, a kit capable of detecting a disease associated with abnormal production of GMP-130 and a kit capable of easily quantifying the amount of GMP-130 in plasma was prepared, thereby completing the present invention.

 Hereinafter, the present invention will be described in detail.

The following diseases associated with abnormal GMP-130 production (1)
Diseases such as (2) and (3).

(1) Diseases due to destruction of cells and organs with relatively high GMP-130 content (2) Diseases due to quantitative abnormalities in cells and organs with relatively high GMP-130 content (3) Comparative disease with GMP-130 content Diseases caused by qualitative abnormalities of many cells and organs.

The above (1), (2), and (3) will be specifically described.
As an example of (1), thrombocytopenic purpura (thrombocytop
enic purpura), systemic lupus erythematosus (systemic l)
upas erythematosus), disseminated intravascular coagulation (disseminat)
Examples of (2) include aplastic anemia, leukemia and thrombocytosis, and examples of (3) include diabetes, arteriosclerosis, renal disease, and thrombosis.

The present inventors have found that GMP-130 having a molecular weight of about 130 kDa, which is lower than that of membrane-bound GMP-140 (molecular weight: 140 kDa), is released and present in human plasma, and thrombocytopenia, leukemia, Diabetes, arteriosclerosis, cerebral infarction, in plasma from patients with renal disease, confirmed that GMP-130 was present in a larger amount than in healthy subjects, and that the amount of GMP-130 was higher than that of diseases with abnormal GMP-130 production. We found that it can be used as an indicator.

Examples of samples that can be used in the present invention include, for example, plasma, hematuria and the like.

In the present invention, the method for measuring the amount of GMP-130 in a sample is not particularly limited, and includes, for example, an immunological method. As an immunological method, there is a method using an antibody. In this case, an immunological measurement method using one or more antibodies can be used. As the antibody, a monoclonal antibody and a polyclonal antibody are used, and it is desirable to use a monoclonal antibody in view of its specificity and affinity. On the other hand, depending on the immunological measurement method, there are an enzyme immunoassay method, a radioimmunoassay method, a latex agglutination method, an immunoturbidimetric method, a fluorescence / luminescence immunoassay method, a Western blotting method, and the like, but sensitivity, simplicity,
An enzyme immunoassay is most desirable in terms of non-radioactivity.

By using an anti-GMP-130 antibody as a kit for measuring the amount of GMP-130, the amount of GMP-130 in the sample can be easily measured. The reagent used in the kit may be in the form of a solution or a lyophilized product.

〔Example〕

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited only to the scope of the following examples.

Example 1 Preparation of Anti-GMP-130 Antibody (1) Preparation of Monoclonal Antibody Against GMP-130 by Platelet Immunization 2 × 10 ⁸ freshly washed platelets were dissolved in 0.1 ml of physiological saline and an equal volume of complete Freund's adjuvant. And emulsified, and injected intraperitoneally into Balb / c mice. Four weeks later, only 2 × 10 ⁸ washed platelets were injected intraperitoneally into the same mice.
Three days later, spleen cells were obtained from spleens removed from the mice, mixed with mouse myeloma cells (SP2 / 0) at a cell ratio of 10: 1, and incubated for 1 minute in the presence of 50% polyethylene glycol and 20% dimethyl sulfoxide. Notify and perform cell fusion. Serum-free DME [Dulbecc's Modified Eagle (Dulbecc
o's Medified Eagle's)]
Remove the supernatant by centrifugation and contain 10% fetal bovine serum
The cells were suspended in a DME medium and dispensed into a 96-well microtiter plate at 2 × 10 ⁴ cells per well. Thereafter, half of the medium was replaced with HAT medium every 1 to 3 days.
0-20 days later, the culture supernatant of the wells where the fused cells (hybridomas) have grown is collected, and the presence or absence of antibody production is determined by ELISA.
First, three hybridomas producing an antibody against GMP-140 were selected.

These hybridomas were cloned twice by the limiting dilution method, and the reactivity of the monoclonal antibodies produced by these hybridomas with GMP-130 was confirmed by Western blotting. As a result, GMP-130
As a clone of a hybridoma producing an anti-GMP-130 antibody which also reacts with the above, one clone strain PL7-6 was obtained. To obtain large quantities of this monoclonal antibody, Balb
/ c mice were intraperitoneally injected with about 2 × 10 ⁷ hybridomas to form ascites tumors, and 10 days later, ascites was collected, and ammonium sulfate was salted out from the ascites by a conventional method, and DEAE cellulose column chromatography was performed. The anti-GMP-130 monoclonal antibody, that is, the PL7-6 monoclonal antibody was purified by lithography. EIA showed PL7-6 monoclonal antibody was IgG
I confirmed that it was one class. This clone strain has been named Hybridoma PL7-6, designated as Hybridoma PL7-6, and deposited with the Microorganisms and Technology Research Institute of the National Institute of Advanced Industrial Science and Technology under the name of Microtechnical Laboratory No. 11073 (FERM P-1073).

(2) Preparation of monoclonal antibody against GMP-130 by soluble antigen immunization (2)-(1) Preparation of washed platelets A platelet fraction was obtained by centrifugation at 700 g for 15 minutes from citrated blood collected from a normal human. After the RCD solution (36m
M citric acid, 5 mM glucose, 1 mM MgCl ₂ , 103 mM NaC
Platelets washed twice by centrifugation twice with l, 20 ng / ml prostaglandin E1) are further washed by an albumin density gradient method, and Ca ²⁺ -free HEPES-Tyrode buffer. The suspension was resuspended in liquid to prepare washed platelets.

(2)-(2) Crude purification of GMP-140 antigen Triton X-100 (Rohm and Haas) surfactant was added to the washed platelets obtained in (2)-(1) so that the final concentration was 1%. The soluble fraction was passed through a Wheat germ agglutinin-immobilized agarose column, and the fraction adsorbed on the column was subjected to 0.1 M glycine-HC.
Elution was carried out with 1 buffer (pH 2.2) to obtain a crudely purified GMP-140 antigen.

(2)-(3) Pass the crude purified GMP-140 antigen through the antibody column on which PL7-6 is immobilized, and elute the fraction adsorbed on the column with 0.1 M glycine-HCl buffer (pH 2.2) And recovered as purified GMP-140 antigen.

(2)-(4) GMP-140 antigen 50 obtained in Example 1- (2)-(3)
μg was dissolved in 0.1 ml of physiological saline, and an equal volume of complete Freund's adjuvant was added to emulsify and injected intraperitoneally into Balb / c mice. Four weeks later, only 50 μg of the antigen was injected intraperitoneally into the same mouse. Three days later, spleen cells were obtained from spleens removed from the mice, mixed with mouse myeloma cells SP2 / 0 at a cell number of 10: 1, and mixed with 50% polyethylene glycol and 20%.
The cell was fused in the presence of 1% dimethyl sulfoxide for 1 minute. After adding and diluting a serum-free DME medium, the supernatant was removed by centrifugation, and DM containing 10% fetal bovine serum was added.
The cells were suspended in E medium and dispensed into a 96-well microtiter plate at 2 × 10 ⁴ cells per well. Thereafter, half of the medium was replaced with HAT medium every 1-3 days,
After about 20 days, the culture supernatant of the well where the hybridoma had grown was collected, and the presence or absence of the antibody-producing ability was examined by ELISA or the like. First, three hybridomas producing an antibody against GMP-140 were selected.

These hybridomas were subjected to limiting dilution by 2
Repeat cloning was performed, and the reactivity of the monoclonal antibodies produced by these hybridomas with GMP-130 was confirmed by Western blotting. As a result, GMP-130
As a hybridoma clone producing an anti-GMP-130 antibody that also reacts with, one strain of clone turnip WGA-1 was obtained. To obtain this monoclonal antibody in large quantities, Ba
Approximately 2 × 10 ⁷ hybridomas were injected into the abdominal cavity of lb / c mouse, and ascites tumor was formed. After 10 days, ascites was collected, and ammonium sulfate was precipitated by a conventional method. The -130 monoclonal antibody, ie, the WGA-1 monoclonal antibody was purified.

It was confirmed by EIA that the WGA-1 monoclonal antibody was of the IgG1 class. This clone strain was named Hybridoma WGA-1 and designated as Hybridoma WGA-1, and deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology as Fermentation No. 11072 (FERM P-11072).

Also, a WGA-1 monoclonal antibody and PL7 of Example 1 were used.
The fact that the -6 monoclonal antibody has a different antigen recognition site to GMP-130 was confirmed by a competitive EIA method.

(3) Preparation of polyclonal antibody against GMP-130 0.5 mg of the purified GMP-140 antigen obtained in Example 1- (2)-(3) was dissolved in 0.5 ml of physiological saline, and an equal amount of complete Freund was added thereto. -After adjuvant was added and emulsified, it was injected subcutaneously into rabbits. Every 2 weeks, the same amount of antigen was emulsified with incomplete Freund's adjuvant and injected subcutaneously four times. The whole blood was collected 10 days after the final immunization, left at room temperature for 60 minutes, and centrifuged. Anti-GMP-140
Antiserum containing the antibody was obtained. An anti-GMP-140 polyclonal antibody was purified from the antiserum using a Protein A column by a conventional method.

The specificity and titer of this antibody were confirmed by a method well-known to those skilled in the art, that is, ELISA, Western blotting, and it was confirmed that the polyclonal antibody sufficiently recognized GMP-130. The following were used as 130 polyclonal antibodies.

Example 2 Isolation of GMP-130 and Partial Identification from Amino Acid Sequence 5 l of normal human plasma was passed through a WGGA-1 antibody column, and the adsorbed fraction was eluted with a 0.1 M glycine-HCl (pH 2.2) buffer. Analysis of this fraction by SDS-PAGE revealed a single band at a molecular weight of 130 kDa. The platelet-derived GMP-140 analyzed at the same time showed a molecular weight of 140 kDa, and the molecular weight was clearly different. A protein purified from this plasma and having a molecular weight of 130 kDa, that is, GMP-130, was produced in Example 1.
Anti-GMP-130 antibodies (WGA1, PL7
-6, polyclonal antibody) and Western blotting showed strong reactivity, and the antigenicities of GMP-130 and GMP-140 were very similar.

Then 40kDa fragment of the 130kDa protein obtained by trypsin digestion, and V ₈ N-terminal amino acid sequence analysis of the 60kDa fragment obtained by protease degradation was performed. As a result, each N-terminal amino acid sequence was AFQHQS and HXLKKKXAL. These are GMP-
140 amino acid sequences 240-245 and 118-126 [Johnston GI et al., Cell, 56, 1033.
(1989)], GMP-130 and GMP-130 have the same amino acid sequence, and their antigenicities are very similar.
Was identified as a free form in the blood.

Example 3 Measurement of the amount of GMP-130 by sandwich EIA using two types of monoclonal antibodies (1) Preparation of monoclonal antibody-bound beads Individual GMP-130 monoclonal antibody WG obtained in Example 1 above
50 ml of 0.1 M phosphate buffer (pH 8.0) containing 1 mg of A-1
Polystyrene balls (Sekisui Chemical Co., particle size 6.35mm) 10
0 cells were added and reacted at 5 ° C. for 16 hours to immobilize the antibody on the beads. After washing the beads with physiological saline, the beads were immersed in phosphate buffered saline containing 1% bovine serum albumin (BSA), and allowed to stand at 5 ° C overnight to obtain anti-GMP-130 monoclonal antibody-bound beads.

(2) Preparation of enzyme-labeled monoclonal antibody GMP-130 monoclonal antibody PL7 obtained in Example 1 above
-6, the peroxidase (Boehringer-Mannheim) was used according to the method of Nakane et al. [Journal of
Histochemistry and cytochemistry (J.Hi
stochem. Cytochem.), Vol. 22, p. 1084 (1987)] to obtain a labeled antibody. That is, 10 mg of peroxidase is dissolved in 2 ml of purified water, and 0.2 ml of 0.1 M potassium periodate is added. After reacting at room temperature for 20 minutes, dialysis is performed overnight at 4 ° C. against 1 mM acetate buffer (pH 9.0). Meanwhile, P
2 mg of L7-6 antibody was added to 1.5 ml of phosphate buffered saline (pH 7.
4) Dissolve in 10 mM carbonate buffer (pH 9.5) at 4 ° C overnight
This was mixed with periodate-treated peroxidase, and allowed to react at room temperature for 2 hours. Then, 0.1 ml of sodium borohydride (4 mg / ml) was added, and the mixture was added at 4 ° C.
After 2 hours, fractionation was performed by gel filtration using Ultrogel AcA22 (manufactured by LKB) equilibrated with phosphate buffered saline (pH 7.4), and fractions having the same peroxidase activity and antibody activity were collected. Final concentration of sodium merthiolate is 0.
It was added so as to be 01%, and stored at 4 ° C.

(3) Measurement of GMP-130 The EIA method was performed as follows. 300 μl of the sample is placed in a tube, and the immobilized antibody beads are placed one by one in the tube, and the first incubation is performed at 37 ° C. for 20 minutes. Next, the beads are washed three times with 3 ml of physiological saline, 300 μl of the labeled antibody solution (300-fold dilution) is placed in the tube containing the beads, and a second incubation is performed at 37 ° C. for 20 minutes.

Next, the beads were washed three times with 3 ml of physiological saline, and the beads were transferred to another tube. The beads were added thereto with a coloring reagent (1 mg / ml of o-phenylenediamine, 0.1 M citrate buffer containing 0.01% of H ₂ O ₂ ). pH5.0) and react at room temperature for 15 minutes to remove 1N H ₂ SO ₄
1 ml was added to stop the reaction. The absorbance at a wavelength of 492 nm was measured.

As the concentration of GMP-130 increases, the absorbance of the color increases,
It became clear that GMP-130 can be measured by this measurement method.

Example 4 GMP-130 by Competitive RIA Using Polyclonal Antibody
Measurement of Amount (1) Preparation of ¹²⁵ I-Labeled GMP-130 Using Enzymo beads manufactured by Bio-Rad (Costrini NY et al., Proceedings of the National Academy)
Of Sciences of the USA, Vol. 76, No. 3242
(1979)].

(2) Measurement of GMP-130 A 0.1 ml sample was placed in a test tube, and 0.1 I of the ¹²⁵ I-labeled antigen solution was added thereto.
Then, 0.1 ml of a first antibody solution (0.1% BSA / TBS containing anti-GMP-130 polyclonal antibody) is added, and the mixture is reacted at room temperature for 2 hours. Add the second antibody solution (3% goat anti-rabbit Ig)
1 ml of G antibody and 0.1% BSA / TBS containing 2.5% PEG-6000 was added, and the mixture was reacted at room temperature for 10 minutes, centrifuged at 1000 g for 20 minutes, the supernatant was discarded, and the radioactivity in the precipitate was measured. Antigen GMP
The radioactivity decreases as the concentration of -130 increases,
It was shown that GMP-130 can be measured by this measurement method.

Example 5 Production of GMP-130 Measurement EIA Kit and GMP-13 in Plasma of Various Patients
Measurement of 0 Amount (1) Production of EIA kit The composition of the EIA kit (one dose) is shown below.

1. One antibody-immobilized bead [The monoclonal antibody WGA-1 produced by Hybridoma Microtechnical Laboratory No. 11072 (FERM P-11072) deposited at the National Institute of Microbial Technology is immobilized. 2. Peroxidase-labeled antibody 0.3 ml [Monoclonal antibody PL7-6 produced by Hybridoma Microtechnical Research Laboratories No. 11073 (FERM P-11073) deposited at the National Institute of Advanced Industrial Science and Technology) 3. Coloring reagent 0.3 ml (10 mg / ml of o-phenylenediamine HCl, hydrogen peroxide 0.1 ml)
Citrate buffer solution containing 01% pH 5.0) 4. Reaction stop solution (1N sulfuric acid) 1.0 ml 5. Standard product (1% B containing 4, 2, 1, 0.5 μg / ml of GMP-130)
(2) Measurement of GMP-130 in patient plasma Using the above EIA kit, 20 healthy subjects, 30 diabetic patients,
5 leukemia patients, 7 arteriosclerosis, 3 thrombocytopenia,
GMP-130 levels were measured based on Example 3 using the plasma of 12 cerebral infarction patients and 3 renal disease patients. As a result of obtaining a calibration curve using a standard product, a calibration curve as shown in FIG. 1 was obtained. In the graph of FIG. 1, the horizontal axis represents the GMP-130 concentration (mg
/ l), and the vertical axis indicates absorbance (A ₄₉₂ nm). Using this calibration curve from the absorbance obtained by measuring the sample, GMP-130
The amount was determined.

FIG. 2 shows the measurement results of GMP-130 in plasma. That is, FIG. 2 is a graph in which the amount of GMP-130 in plasma measured according to the present invention is plotted for each case.

As shown in FIG. 2, there was a difference in the distribution of GMP-130 in the plasma of healthy subjects and diabetic patients, leukemia patients, arteriosclerosis, thrombocytopenia, cerebral infarction and renal disease patients. That is, it was found that the amount of GMP-130 in plasma was higher in the above patients than in healthy individuals.

The plasma sample from the patient was subjected to ultracentrifugation (10
(5,000 g, 60 minutes), the supernatant was measured as a sample, and a value almost similar to that in FIG. 2 was obtained. In the patient plasma, GMP-130, but not membrane-bound GMP-140, was increased. It was confirmed that.

Example 6 Production of GMP-130 Measurement RIA Kit and GMP-13 in Various Patient Plasma
Measurement of 0 Amount (1) Production of RIA Kit The composition of the RIA kit (one dose) is shown below.

1. ¹²⁵ I labeled antigen solution 0.1 ml (0.1% BSA / T containing 20000 cpm of ¹²⁵ I labeled GMP-130
BS) 2. First antibody solution 0.1 ml (0.1% BSA / TBS containing rabbit anti-GMP-130 antibody) 3. Second antibody solution 1 ml (containing 3% goat anti-rabbit IgG antibody and 2.5% PEG-6000) 0.1% BSA / TBS) 4. Standard solution 0.1 ml (1% B containing 4, 2, 1, 0.5 μg / ml of GMP-130 antigen)
(2) Measurement of GMP-130 in Patient Plasma Using the RIA kit described above, the same samples as in Example 5 were measured by the method of Example 4 and the amount of GMP-130 in each sample was determined as in Example. Almost the same value as 5 was obtained.

〔The invention's effect〕

As described in detail above, the present invention
It has been found that the amount of GMP-130 is a marker for a disease associated with abnormal GMP-130 production, such as thrombocytopenia, diabetes, leukemia, arteriosclerosis, etc., and a new detection method and quantification of the above-mentioned disease. Kit was provided.

[Brief description of the drawings]

FIG. 1 is a graph showing a calibration curve used in the kit for detecting GMP-130 in Example 5 of the present invention, and FIG. 2 is a view showing the results of measuring the concentration of GMP-130 in plasma measured using the kit. It is.

────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu-shi, Shiga Central Research Laboratory of Takara Shuzo Co., Ltd. (58) Fields investigated (Int. Cl. ⁶ , DB name) ) G01N 33/53 G01N 33/68 G01N 33/50

Claims (3)

(57) [Claims] 1. GMP-130 having the following physicochemical properties: (B) The molecular weight analyzed by the SDS-PAGE method is 130 kDa. (B) A 40 kDa fragment was obtained by trypsin digestion, and the N-terminal amino acid sequence was AFQHQS. (C) fragments of 60kDa was obtained by V ₈ protease degradation, the N-terminal amino acid sequence is, HXLKKKXA
L. 2. The method according to claim 1, which is in plasma.
(C) A method for detecting a disease associated with abnormal GMP-130 production, wherein the amount of GMP-130 having physicochemical properties is compared with a normal value. 3. A kit for detecting a disease associated with abnormal GMP-130 production, which has the physicochemical properties of (a) to (c) according to claim 1, wherein the kit comprises an anti-GMP-130 antibody. Characteristic kit for detecting diseases associated with abnormal production of GMP-130