JP2003107088A - Disseminated intravascular coagulation syndrome and method for detecting prestage of its crisis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for highly sensitively detecting the onset of the disseminated intravascular coagulation (DIC) syndrome and especially the prestages (pre-pre DCI stage and pre-DCI stage) of the crisis of DIC. SOLUTION: The concentration of nick β2 glycoprotein I in a humor sample is measured to detect the disseminated intravascular coagulation syndrome or the prestages of its onset. Or the concentration (N) of nick β2 glycoprotein I and the concentration (T) of total β2 glycoprotein I in the humor sample are measured to compute the ratio (N/T) of the concentration (N) of nick β2 glycoprotein I to the concentration (T) of total β2 glycoprotein I, and the detection is performed with the ratio as an index.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a disseminated intravascular coagulation syndrome.
The present invention relates to a method for detecting (Culual Coagulation: DIC) or a pre-symptomatic stage thereof, and in particular, it can be used for early prediction of DIC onset.

[0002] In the present specification, "nick β2 glycoprotein I" is composed of two polypeptide chains which are cleaved by a part of an amino acid sequence by a protease, but are bound by a disulfide bond to β2.
Means Glycoprotein I. In the present specification, when it is necessary to distinguish β2 glycoprotein I that has not been cleaved by protease from the above-mentioned “nick β2 glycoprotein I”, “intact β2
Sometimes referred to as "Glycoprotein I". Therefore, in the present specification, when simply referred to as “β2 glycoprotein I”, “intact β2” is used unless otherwise specified.
Shall refer to Glycoprotein I ". Further, in the present specification, the “total β2 glycoprotein I” includes both the “nick β2 glycoprotein I” and the “intact β2 glycoprotein I”.

[0003]

BACKGROUND OF THE INVENTION β2 Glycoprotein I is a glycoprotein present in the blood of healthy people at a concentration of about 200 μg / mL, has the property of binding negatively charged phospholipids, and has a contact phase of endogenous coagulation, It is known to exhibit anticoagulant activity by suppressing platelet aggregation by ADP, activating factor 5 and phospholipid-dependent prothrombinase activity, and interaction between protein S and its binding protein. In addition, in nick β2 glycoprotein I in which a part of β2 glycoprotein I was cleaved by a protease, the affinity for the negatively charged phospholipid was that of intact β2 glycoprotein I.
It has been reported that the affinity is decreased to about 1/100 or less [J. E. Hunt et al., Proc. Natl.
Acad. Sci. USA, vol. 90, p. 2141-
2145, (1993)]. In various pathological conditions, when the intravascular coagulation system is activated, the fibrinolytic enzyme plasmin is subsequently activated, so that β2 glycoprotein I is cleaved by this plasmin [Okura et al., Blood, 91, p. 4173 to p. 4179, (1998)], and it is considered that nick β2 glycoprotein I is released into the blood due to a decrease in affinity with negatively charged phospholipids. In fact, it has been found that blood nick β2 glycoprotein I levels are increased in patients with leukemia and antiphospholipid antibody syndrome [ItohY et al., J. Am. Bioche
m. , Volume 128, pages 1017 to 1024 (2000
)], Many of these patients have abnormal coagulation and fibrinolysis in vivo.

Disseminated intravascular coagulation (DIC), which is caused by a serious underlying disease such as malignant tumor, leukemia, or severe infection, causes bleeding tendency due to coagulative consumption and organ damage due to microvascular thrombosis. It is characterized by a very high mortality syndrome and requires early diagnostic treatment to maintain the patient's life and improve prognosis. Therefore, there is a demand for a molecular marker that reflects the coagulation-fibrinolytic system enhanced state at the earliest possible stage. Conventionally, for the diagnosis of DIC, various diagnostic markers established by the Ministry of Health, Labor and Welfare, various coagulation fibrinolytic system molecular markers [eg soluble fibrin monomer (SF), DD
Dimer (DD), thrombin / antithrombin III
The complex (TAT), plasmin / α2 plasmin inhibitor complex (PIC)] and the like are measured in clinical practice, but the diagnosis of DIC, especially its early diagnosis, that is, the pre-symptomatic stage (pre-pre-DIC and pre-DIC) ) Was insufficient in sensitivity to detect.

[0005]

DISCLOSURE OF THE INVENTION The inventors of the present invention have earnestly studied for the purpose of developing a method capable of detecting the preclinical stage of DIC with high sensitivity, and found that nick β2 glycoprotein I
Was found to be effective as a highly sensitive molecular marker for the onset of DIC and its pre-onset stage. It was also found that the ratio of the nick β2 glycoprotein I concentration to the total β2 glycoprotein I concentration is also effective for highly sensitive detection of the onset of DIC and its pre-onset stage. The present invention is based on these findings.

[0006]

The present invention relates to a method for detecting disseminated intravascular coagulation or its pre-onset stage, which comprises measuring the concentration of nick β2 glycoprotein I in a body fluid sample. The present invention also measures the concentration (N) of nick β2 glycoprotein I and the concentration (T) of total β2 glycoprotein I in a body fluid sample,
Ratio of concentration (N) of nick β2 glycoprotein I to concentration (T) of total β2 glycoprotein I (N
/ T) and using the ratio as an index to detect a disseminated intravascular coagulation syndrome or a pre-onset stage thereof. Furthermore, in a preferred aspect of the present invention, the above-mentioned concentration measurement is performed based on an immunological assay method or a biochemical assay method.

[0007] In the present specification, the "nick β2 glycoprotein I" is composed of two polypeptide chains which are cleaved by a protease at a part of the amino acid sequence as described above, but are bound by a disulfide bond. Β2 glycoprotein I. Specifically, the “nick β2 glycoprotein I” includes, for example, (1) cleavage at the V domain by plasmin (in human β2 glycoprotein I, the 317th lysine residue and the 318th threonine residue). Nick β2 glycoprotein I that has been cleaved with the residue, and (2) cleavage into the V domain by granulocyte elastase (in human β2 glycoprotein I, the 314th alanine residue and the 315th residue) Nicked β2 glycoprotein I which has been cleaved with the phenylalanine residue of P.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION According to the method of the present invention, disseminated intravascular coagulation (DIC) or its pre-onset stage can be detected with high sensitivity. Here, the DIC is, for example,
It is a state where the judgment is 7 points or more in the "DIC diagnostic criteria" established by the Ministry of Health, Labor and Welfare. In the present specification, DIC
The "pre-symptomatic stage" of is, for example, pre-DIC (pre-
DIC) and pre-pre-DIC (pre-pre-DI)
Means C). Here, the pre-DIC does not reach the state (DIC onset) where the judgment is 7 points or more in the "DIC diagnostic criteria" defined by the Ministry of Health, Labor and Welfare, but, for example,
It refers to a state in which movement is seen in an index that indicates the state of hypercoagulability. Further, the pre-pre-DIC means the above-mentioned pre-DIC.
Although it does not reach the pre-DIC state, it means a state several days before the pre-DIC state (for example, 2-3 days before).

In the method of the present invention, any body fluid sample of any mammal (particularly human) can be used. Examples of the body fluid sample include blood samples, particularly plasma and serum, and it is particularly preferable to use plasma.

The inventor of the present invention, as shown in the examples described later, has a DIC patient, a pre-DIC patient, and a pre-pre-DIC.
It was found that the concentration of nick β2 glycoprotein I contained in the body fluid sample was statistically significantly higher in patients than in healthy subjects. Therefore, in the method of the present invention, DIC or its pre-onset stage can be detected by measuring the concentration of nick β2 glycoprotein I contained in the body fluid sample collected from the test subject. The concentration of nick β2 glycoprotein I can be determined based on, for example, an immunological assay method or a biochemical assay method.

Further, the inventor of the present invention, as shown in Examples described later, has a DIC patient, a pre-DIC patient and a pre-pre-D patient.
In IC patients, the ratio (N) of the concentration (N) of nick β2 glycoprotein I to the concentration (T) of total β2 glycoprotein I in the body fluid sample was higher than that in healthy subjects (N).
/ T) (ie, the R value) was found to be statistically significantly higher. Therefore, in the method of the present invention, the nick β contained in the body fluid sample collected from the test subject
The concentration (N) of 2 glycoprotein I and the concentration (T) of total β2 glycoprotein I were measured, and total β2
By calculating the ratio (N / T) of the concentration (N) of nick β2 glycoprotein I to the concentration (T) of glycoprotein I (that is, R value), and using the R value as an index, DIC or its The pre-symptomatic stage can be detected. The concentration of nick β2 glycoprotein I can be determined based on, for example, the above-mentioned immunological assay method or biochemical assay method, and total β2 glycoprotein I can be determined.
Similarly, the concentration of can be determined based on an immunological assay method or a biochemical assay method.

The above-mentioned immunological measurement of nick β2 glycoprotein I is carried out by, for example, the method described in Japanese Unexamined Patent Publication No. 2000-28607, "A monoclonal that does not react with intact β2 glycoprotein I but reacts with nick β2 glycoprotein I. Antibodies "(hereinafter sometimes referred to as anti-nick GPI monoclonal antibody) or antibody fragments thereof can be used. The anti-nick G
The PI monoclonal antibody is preferably (1)
It does not react with intact β2 glycoprotein I, reacts with nick β2 glycoprotein I, and
Reacts with the V domain of 2 glycoprotein I [especially has an epitope in the region consisting of amino acid residues 242 to 326 (counting from the amino terminal side) in human nick β2 glycoprotein I] ] Monoclonal antibody, more preferably, hybridoma NGPI-59 (FERM P-16892)
Secreted from NGPI-59 or (2) not reacting with intact β2 glycoprotein I, but reacting with nick β2 glycoprotein I, and the domain I of nick β2 glycoprotein I ~
Monoclonal monoclonal antibody that reacts with the region consisting of the IV domain [especially has an epitope in the region consisting of the 1st amino acid residue (counting from the amino terminal side) to the 241st amino acid residue in human nick β2 glycoprotein I] Antibody, more preferably hybridoma NGPI
Mention may be made of the monoclonal antibody NGPI-60 secreted from -60 (FERM P-16893).

The above-mentioned immunological measurement of total β2 glycoprotein I is carried out, for example, by "A monoclonal antibody which reacts with intact β2 glycoprotein I and also with nick β2 glycoprotein I described in JP-A-2000-28607. (Hereinafter, sometimes referred to as anti-total GPI monoclonal antibody) or an antibody fragment thereof. As the anti-total GPI monoclonal antibody, intact β
2 glycoprotein I (particularly human intact β2 glycoprotein I), and nick β2 glycoprotein I [particularly human nick β cleaved at the V domain
2 glycoprotein I], preferably a region consisting of intact β2 glycoprotein I and I domain to IV domain of nick 2 glycoprotein I [eg, in human nick β2 glycoprotein I (counting from the amino terminal side) 1st amino acid residue to 24th
Monoclonal antibody having an epitope in the region consisting of the first amino acid residue] is preferred, and hybridoma N
More preferred is the monoclonal antibody NGPI-23 secreted from GPI-23 (FERM P-16891).

As the antibody fragment of the anti-nick GPI monoclonal antibody or the anti-total GPI monoclonal antibody, an antibody fragment which is a fragment of each of the above-mentioned monoclonal antibodies and has the same reaction specificity as the original monoclonal antibody is used. be able to. Antibody fragments include, for example, Fab, Fa
b ', F (ab') ₂ or Fv is included.

An immunological assay for nick β2 glycoprotein I is carried out, for example, by immobilizing the above-mentioned anti-nick GPI monoclonal antibody or an antibody fragment thereof on an insoluble carrier as the first antibody, and then immobilizing the immobilized first antibody and body fluid. A sample is brought into contact with the sample, and subsequently, a second antibody obtained by labeling the above-mentioned anti-nick GPI monoclonal antibody or antibody fragment thereof different from the first antibody, or the above-mentioned anti-total GPI monoclonal antibody or antibody fragment thereof is labeled. The second antibody bound to the immobilized first antibody-nick β2 glycoprotein I complex or the immobilized first antibody-nick β2 glycoprotein I complex when brought into contact with the second antibody labeled with. Since it is possible to detect the signal from the label of the second antibody that has not bound, it is possible to detect the signal in the body fluid sample. Tsu can measure the amount of click β2 glycoprotein I (sandwich method).

Further, at least one kind of the above-mentioned anti-nick GPI monoclonal antibody or an antibody fragment thereof,
An anti-nick GPI monoclonal antibody or an antibody fragment thereof or another anti-total GPI monoclonal antibody or an antibody fragment thereof of a different type is immobilized on an insoluble carrier, and the immobilized monoclonal antibody or antibody fragment thereof and a body fluid sample are prepared. Contact with the intact β2 glycoprotein I in the body fluid sample does not cause an agglutination reaction, and the agglutination reaction can occur only with the nick β2 glycoprotein I. I
Can be measured (aggregation method).

Therefore, in the above-described immunological analysis method for nick β2 glycoprotein I, the body fluid sample is not pretreated (for example, the intact β2 glycoprotein I and the nick β2 glycoprotein I are preliminarily obtained by a chromatographic method). Even if they are used as they are without separation operation), interference of intact β2 glycoprotein I present in the body fluid sample can be avoided.

In the immunological analysis method of nick β2 glycoprotein I using the sandwich method, specifically,
The anti-nick GPI monoclonal antibody (for example, the above-mentioned monoclonal antibody NGPI-59 or monoclonal antibody NGPI-60) or an antibody fragment thereof is immobilized on a suitable insoluble carrier (first antibody). Next, in order to avoid non-specific binding between the insoluble carrier and the body fluid sample,
The surface of the insoluble carrier is coated with a suitable blocking agent [eg, bovine serum albumin (BSA), gelatin, etc.]. Subsequently, an undiluted body fluid sample is added to the mixture for a certain period of time (for example, 5 minutes to 3 hours) and a certain temperature (for example, 4 ° C to 4 ° C)
Contact at 0 ° C., preferably near room temperature to react (1
Next reaction). Subsequently, a second antibody obtained by labeling the above-mentioned anti-nick GPI monoclonal antibody or an antibody fragment thereof different from the monoclonal antibody used as the primary antibody, or the above-mentioned anti-total GPI monoclonal antibody (for example, a monoclonal antibody). NGPI-23)
Alternatively, a labeled second antibody is added to the antibody fragment and contacted and reacted for a certain period of time (for example, 5 minutes to 3 hours) and a certain temperature (for example, 4 ° C. to 40 ° C., preferably around room temperature) (2 Next reaction). This is washed with an appropriate washing solution (for example, physiological saline containing a surfactant), and then the amount of the labeled antibody present on the insoluble carrier is quantified.
From that value, the amount of nick β2 glycoprotein I in the body fluid sample can be calculated. It is also possible to carry out the primary reaction and the secondary reaction at the same time.

The immunological assay for total β2 glycoprotein I is carried out, for example, in the assay for nick β2 glycoprotein I described above, in place of the anti-nick β2GPI monoclonal antibody or its antibody fragment. It is possible to measure by a similar method using the fragment. (Sandwich method).

The insoluble carrier that can be used in the immunological analysis method by the sandwich method is not particularly limited, and examples thereof include polyethylene, polystyrene,
Polymers such as polypropylene, polyvinyl chloride, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, and polysaccharides, other nitrocellulose,
Examples include paper, agarose, and combinations thereof. It is advantageous to use an enzyme, a fluorescent substance, or a luminescent substance as the labeling substance. Examples of the enzyme include alkaline phosphatase, peroxidase, β
-D-galactosidase and the like, and as a fluorescent substance,
For example, fluorescein isothiocyanate, etc.,
Examples of the luminescent substance include acridinium ester,
Luciferin and the like can be used.

In the immunological analysis method utilizing the agglutination reaction, as the insoluble carrier, any insoluble carrier generally used in the immunological analysis method utilizing the agglutination reaction of the antigen-antibody reaction can be used. Latex particles (particularly polystyrene latex particles) can be mentioned. To immobilize the monoclonal antibody on the insoluble carrier, a known method, for example, a chemical bonding method (using carbodiimide, glutaraldehyde or the like as a cross-linking agent) or a physical adsorption method can be used.

The concentrations of the above-mentioned nick β2 glycoprotein I and total β2 glycoprotein I can also be measured biochemically, for example. For example, the biochemical assay method for nick β2 glycoprotein I and intact β2 glycoprotein I is as follows.
It can be carried out by fractionation by affinity chromatography using a HiTrap-Heparin column [Okura et al., Blood, No. 91.
Vol., Pp. 4173 to 4179, (1998)].
At this time, the nick β2 glycoprotein I and the intact β2 glycoprotein I can be separated due to the difference in their elution positions (that is, due to the difference in affinity for heparin). By quantifying the protein in the eluted fraction, the amounts of nick β2 glycoprotein I and intact β2 glycoprotein I can be measured. The above-mentioned nick β2 glycoprotein I and intact β2 glycoprotein I
The total β2 glycoprotein I concentration is calculated from the respective amounts, and the ratio (N / T = R) of the nick β2 glycoprotein I concentration (N) to the total β2 glycoprotein I concentration (T) is calculated from these values. be able to.

[0023]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention.

Example 1 << Measurement of Nick β2 Glycoprotein I >>
In this example, a pre-pre-DIC patient, a pre-DIC patient,
Β2 in the DIC and DIC patient groups, and in the healthy subject group
Glycoprotein I concentration was measured. Here, each patient was diagnosed according to the following criteria. DIC patients: Diagnosis according to DIC diagnostic criteria of the Ministry of Health, Labor and Welfare. Pre-DIC patient: A patient who does not meet the DIC diagnostic criteria of the Ministry of Health, Labor and Welfare but has a movement that is an index of hypercoagulable state. Pre-pre-DIC patient: A patient with a condition of several days before the pre-DIC (2 to 3 days before).

Nickel β was prepared by the following method using a plasma sample collected from each patient and a plasma sample collected from a healthy person who were diagnosed by the above-mentioned conventional diagnostic methods as samples.
2 Glycoprotein I was measured. Fragment F (ab ') ₂ of monoclonal antibody NGPI-60 was added at 10 μg /
Tris buffer A containing at a concentration of mL [50 mmol /
L Tris-HCl, 0.15 mol / L NaCl
(PH 7.5)] 100 μL was added to each well of a 96-well ELISA microtiter plate (Immulon-II; Nippon Dynatec Co., Ltd.), and 1 at 4 ° C.
It was left for 8 hours. Wash the plate with washing solution W (0.05%
Tween-20-0.5 mol / L NaCl) 3
Washed twice. To the wells of the plate thus sensitized with the antibody, 200 ng of nick β2 glycoprotein I was added.
/ ML, 100 ng / mL, 50 ng / mL, 25 ng
/ ML, 12.5 ng / mL, and 6.25 ng / mL concentrations of Tris buffer B [20 mmol / L
Tris-HCl, 0.5 mol / L NaCl, 0.
05% Tween-20 (pH 7.6)], or 100 μL of the standard sample prepared by adding 5 μL of the sample plasma diluted with the same Tris buffer B to 100 μL, and Reacted for hours.

Next, the cleaning liquid W (0.05% Twee)
(n-20-0.5 mol / L NaCl) three times, and then biotin-labeled monoclonal antibody NGPI-2
Tris buffer B [20 mmol / L Tris-HC containing the fragment F (ab ′) ₂ of 3 in an amount of 2 μg / mL.
1, 0.5 mol / L NaCl, 0.05% Twee
n-20 (pH 7.6)] 100 μL was added, and the mixture was reacted at 25 ° C. for 1 hour. Then, the cleaning liquid W (0.05%
Tween-20-0.5 mol / L NaCl) 3
After washing twice, 100 μL of peroxidase-labeled avidin (Dako Co.) diluted 2000 times with Tris buffer B
Was added and reacted at 25 ° C. for 1 hour. 3 with the cleaning liquid W
After washing twice, the enzyme substrate solution [10 mmol / L phenol / 0.5 mmol / L 4-aminoantipyrine /
50 mmol / L Tr containing 0.005% hydrogen peroxide
is-HCl, 0.15 mol / L NaCl (pH
7.5)] was added to each well in an amount of 200 μL, and the absorbance of each well at 492 nm was measured with a microplate reader (MPR A4i type; Tosoh Corporation). A calibration curve was prepared based on the absorbance of the standard sample at each concentration, and the concentration of nick β2 glycoprotein I in the sample plasma was determined from this calibration curve.

The results are shown in Table 1 and FIG. The mean value ± SD of the nick β2 glycoprotein I concentration in each group was 58.60 ± 38.98 ng / mL in the healthy subject group (44 cases) and 131.14 ± 39.0 in the pre-pre-DIC group (109 cases).
2 ng / mL, pre-DIC group (47 cases) 125.52
± 39.89 ng / mL, and DIC patient group (33 cases)
Was 111.67 ± 51.02 ng / mL. The pre-pre-DIC, pre-DIC and DIC patient groups were p <0.001 with respect to the healthy subjects, and a statistically significant difference was observed. As described above, also in the pre-pre-DIC group, the nick β2 glycoprotein I concentration in plasma was already significantly higher than that in the healthy person.

<< Table 1 >> Pre-DIC Pre-DIC Pre-DIC DIC concentration (ng / mL) 58.60 ± 38.98 131.14 ± 39.02 125.52 ± 39.89 111.67 ± 51.02 Statistical significance: Pre-pre-DIC vs DIC: p <0.05 Normal subjects vs pre-pre-DIC: p <0.001 healthy person vs pre-pre-DIC: p <0.001 healthy person vs DIC: p <0.001

[0028]

Example 2 << Total β2 Glycoprotein I and R
Measurement of Value >> In this embodiment, pre-pre-DIC, pre-DIC
And DIC patient group, and total β in healthy subject group
2 Glycoprotein I was measured and R value was calculated. The same plasma sample as in Example 1 was used as a sample, and total β2 glycoprotein I was measured by the following method. Tris buffer A [50 mmol / L Tri containing the monoclonal antibody NGPI-23 at a concentration of 5 μg / mL
s-HCl, 0.15 mol / L NaCl (pH 7.
5)] 50 μL was placed in each well of a 96-well ELISA microtiter plate (Immulon-II; Nippon Dynatec Co., Ltd.) and left at 4 ° C. for 18 hours. The plate was washed with the washing solution W (0.05% Tween).
(n-20-0.5 mol / L NaCl) 3 times. Into the wells of the plate thus sensitized with the antibody, 200 ng of intact β2 glycoprotein I was added.
mL, 100 ng / mL, 50 ng / mL, 25 ng /
mL, 12.5 ng / mL and 6.25 ng / mL concentrations of Tris buffer B [20 mmol / LTr
is-HCl, 0.5 mol / L NaCl, 0.05
% Tween-20 (pH 7.6)] and 50 μL of the standard sample prepared by adding each sample to the sample, and 50 μL of the sample sample obtained by diluting the sample plasma 8000 times with the same Tris buffer B, and reacting at 25 ° C. for 2 hours. Let

Next, the cleaning liquid W (0.05% Twee)
(n-20-0.5 mol / L NaCl), and washed with biotin-labeled anti-human β2 glycoprotein I three times.
Rabbit polyclonal antibody (Sedalene) 2.5μ
Tris buffer B [20 mmol contained in an amount of g / mL]
/ L Tris-HCl, 0.5 mol / L NaC
1, 0.05% Tween-20 (pH 7.6)] 50
μL was added, and the mixture was reacted at 25 ° C. for 1 hour. Then, the cleaning liquid W (0.05% Tween-20-0.5 mol
/ L NaCl) three times, and then 100 μL of peroxidase-labeled avidin (Dako Co.) diluted 2000 times with Tris buffer B was added and reacted at 25 ° C. for 1 hour. After washing three times with the washing solution W, the enzyme substrate solution [10
mmol / L phenol / 0.5 mmol / L 4-aminoantipyrine / 0.005% hydrogen peroxide-containing Tris buffer A [50 mmol / L Tris-HCl,
0.15 mol / L NaCl (pH 7.5)]] was added to each well in an amount of 200 μL, and the absorbance at 492 nm of each well was measured using a microplate reader (MPRA A).
4i type; Tosoh). A calibration curve was prepared based on the absorbance of the standard sample at each concentration, and the total β2 glycoprotein I concentration in the sample plasma was determined from this calibration curve.

Using the nick β2 glycoprotein I concentration (N) measured in Example 1 above, the ratio of the nick β2 glycoprotein I concentration (N) to the total β2 glycoprotein I concentration (T) measured in this Example (N / T
= R) was calculated for individual specimens. The results are shown in Table 2.
And shown in FIG. In the DIC patient group, the pre-pre-DIC group, and the pre-DIC group, p <0.001 was obtained, and a statistically significant difference was observed with respect to the healthy subjects. As described above, it was confirmed that the R value in the pre-pre-DIC group was already significantly higher than that in the healthy person.

<< Table 2 >> Healthy person Pre-pre DIC Pre-DIC DIC R 0.44 ± 0.22 1.35 ± 1.16 1.49 ± 0.99 2.30 ± 1.79 Statistical significance: Healthy person vs Pre-DIC: p <0.001 Healthy person vs Pre-pre-DIC: p <0.001 DIC vs healthy person: p <0.001 DIC vs pre-DIC: p <0.02 DIC vs pre-pre DIC: p = 0.005 pre-DIC vs pre-pre DIC: No significant difference

[0032]

Example 3 << TAT, PIC, SF and DD measurement values and significant difference test between each sample group >> In this example, the pre-pre-DIC, the pre-DIC and DIC patient group and the healthy subject group were subjected to TAT ( Thrombin-antithrombin III complex) value, PIC (plasmin
α2 plasmin inhibitor complex) value, DD (DD
The dimer) value and SF (soluble fibrin) value were measured, and the significant difference between each sample group was tested. In this example as well, the same plasma sample used in Examples 1 and 2 was used.
The measurement of the coagulation / fibrinolytic system marker was carried out by ELISA (International Reagent) for TAT, PIC, and DD, and S
For F, the aggregation method (Yatron) was used.

The results are shown in Tables 3 and 4 and FIGS. P <in TAT between healthy people and pre-pre DIC
The value was 0.01, indicating a significant difference. However, none of the other markers was significantly different from that of healthy subjects at the pre-pre-DIC stage. On the other hand, as shown in Examples 1 and 2, p <0.001 in nick β2 glycoprotein I and R value,
A significant difference is recognized. From this, nick β2 glycoprotein I seems to be a marker with higher sensitivity in the pre-onset stage of DIC, as compared with conventional markers for coagulation and fibrinolysis.

<Table 3> Healthy person Pre-pre DIC Pre-DIC DIC TIC (ng / mL) 1.01 ± 1.44 10.67 ± 20.84 15.83 ± 19.38 38.37 ± 28.98 PIC (μg / mL) 0.27 ± 0.35 1.08 ± 0.7 2.20 ± 2.40 5.77 ± 4.37 DD (μg / mL) 0.41 ± 0.39 0.94 ± 1.08 6.76 ± 5.55 17.94 ± 9.96 SF (μg / mL) 3.45 ± 2.06 33.75 ± 10.71 193.77 ± 453.60 353.07 ± 158.10

[Table 4] Healthy vs vs Pre-DIC Healthy vs vs DIC Healthy vs vs DIC TAT p <0.01 p = 0.1499 p <0.001 PIC p <0.05 p <0.001 p <0.001 DD NS p <0.001 p <0.001 SF p = 0.045 p = 0.007 p = 0.0611

[0036]

According to the method of the present invention, the onset of DIC, and particularly the pre-onset stage of DIC (pre-pre-DIC and pre-DIC) can be detected with high sensitivity.

[Brief description of drawings]

FIG. 1 shows the value of nick β2 glycoprotein I (nick β2GPI) concentration in a plasma sample measured by a sandwich enzyme immunoassay according to the method of the present invention.
It is a bar graph shown according to C patient group.

FIG. 2 shows the ratio R calculated from the nick β2 glycoprotein I concentration and the total β2 glycoprotein I concentration in the plasma sample measured and measured by the sandwich enzyme immunoassay according to the method of the present invention, for healthy and DIC patient groups. It is a bar graph.

FIG. 3 is a bar graph showing the TAT concentration in a plasma sample measured by a conventional method for each group of healthy subjects and DIC patients.

FIG. 4 is a bar graph showing the PIC concentration in a plasma sample measured by a conventional method for each group of healthy subjects and DIC patients.

FIG. 5 is a bar graph showing SF concentrations in a plasma sample measured by a conventional method for each group of healthy subjects and DIC patients.

FIG. 6 is a bar graph showing the DD concentration in a plasma sample measured by a conventional method for each group of healthy subjects and DIC patients.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Ieko Masahiro             Ainosato 1-Chome 3-chome, Kita-ku, Sapporo-shi, Hokkaido             3-1308 (72) Inventor Hideo Wada             2213 Honmachi, Matsuzaka City, Mie Prefecture (72) Inventor Hisao Kato             8-13-1013 Kamiyamada, Suita City, Osaka Prefecture (72) Inventor Hideyuki Tanaka             1-11-4 Higashi-Kanda, Chiyoda-ku, Tokyo Stock             In ceremony company Yatron (72) Inventor Yumiko Kazehaya             1-11-4 Higashi-Kanda, Chiyoda-ku, Tokyo Stock             In ceremony company Yatron F-term (reference) 2G045 AA25 CA26 DA77 FB03

Claims (3)

[Claims] 1. A method for detecting disseminated intravascular coagulation syndrome or its pre-onset stage, which comprises measuring the concentration of nick β2 glycoprotein I in a body fluid sample. 2. The concentration (N) of nick β2 glycoprotein I and total β2 glycoprotein I in a body fluid sample.
Concentration (T) of nick β2 glycoprotein I against total β2 glycoprotein I concentration (T)
The method according to claim 1, wherein the ratio (N / T) of the concentrations (N) of the is calculated, and the ratio is used as an index. 3. The method according to claim 1, wherein the concentration is measured based on an immunological assay method or a biochemical assay method.