JP2000111556A - Judgement method of disease condition of rheumatic disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately measure the disease activity of rheumatic disease by sampling blood in a measurement receptacle to allow it to react with a material producing a physiologically active substance in the blood by contacting it with the blood, and measuring a quantity of the physiologic active substance. SOLUTION: Endotoxin is preferable due to high induction activity as a material inducing production of a physiologically active substance, and concentration of the endotoxin in the whole blood in the case of contact with blood is set at about 0.6-100,000 EU. The producing physiologically active substances are a tumor necrosis factor α, a tumor necrosis factor β, and interleukin. Endotoxin free water in the same quantity as sampled blood amount is obtained in a measurement receptacle, and stirred for about an hour at about 37 deg.C and extracted to set the content of endotoxin in the extracted liquid to about 0.5 EU/ml or less. Induction of cytokine is caused conspicuously when the content of endotoxin in the receptacle exceeds 0.5 EU/ml. Quantity of physiologically active substance produced in the blood is determined by oxygen immunoassay and then state of rheumatoid disease is judged thereby.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for judging the condition of a rheumatic disease.

[0002]

2. Description of the Related Art Rheumatoid arthritis patients in Japan
It is said that the ratio is about 3 per 1,000 population, and it is known that the ratio is particularly large for women. (1) Definition: The definition of rheumatism is "a disease that complains of pain and stiffness in motor organs such as bones, joints, muscles, and tendons."
Particularly, rheumatoid arthritis (RA) has polyarthritis as a main symptom, but causes a wide range of lesions in the internal organs, and is considered to be a systemic disease. (2) Etiology: The etiology of rheumatism is determined by the rheumatoid factor (R
Although it is recognized that F) has a strong color like an autoimmune disease such as the presence of F), it is difficult to explain everything with this, and at present it has not yet been elucidated. In addition to this, there are theories of infection, endocrine disorders, and theories of genetics. (3) Diagnosis and treatment: For the diagnosis of rheumatism, 11 diagnostic criteria created in 1958 by the American College of Rheumatology (ARA) were used (Bulletine on
the Rheumatic Diseases, Vol. 9, 175 pages, 1
958), revised in 1987, and the diagnostic criteria
Items (Arthritis and Rheumatism, separate volume,
45, 1987). 1) Morning stiffness lasting at least 1 hour (6 weeks or more) 2) Swelling of 3 or more joints (6 weeks or more) 3) Hand (WRIST), metacarpal (MCP), proximal finger (PIP) )
4) Symmetric joint swelling 5) Changes in X-ray images of hands and fingers 6) Subcutaneous nodules (rheumatic nodules) 7) Presence of RF At least 4 lesions among the above 7 If so, the patient is diagnosed with rheumatism.

In particular, among these criteria, for objective diagnosis of rheumatism, measurement of RF is generally performed. That is, detection of RF is indispensable in diagnosing RA, and is also used for discrimination from other diseases having many joint symptoms (joint pain, arthritis) in addition to RA. However, in fact, there are about several to 10% of RF-positive persons among healthy persons, while those who are diagnosed with rheumatism have an RF-positive rate of 40 to 90%.
6, 1442, 1988). In addition, as a disease in which RF is observed in serum, in addition to RA, rheumatism-related diseases and collagen disease other than RA (Sjogren's syndrome, adjuvant disease, collagen disease, etc.) show a positive rate of 20 to 40%. , Other liver diseases (especially cirrhosis, chronic hepatitis)
And heart disease (especially after a myocardial infarction attack, subacute bacterial endocarditis) show a high positive rate of 30 to 50%. In the treatment of rheumatoid arthritis, various non-steroidal anti-inflammatory drugs are usually most often used.
The F value often remains almost unchanged in relation to the present drug. It has also been reported that administration of corticosteroids, gold preparations, penicillamines, and the like resulted in a decrease in RF antibody titer and a negative response. However, in practice, RF is not used to judge the active stage or therapeutic effect of RA, and the current state is that CRP and erythrocyte are used as indices (Medical Technology, Vol. 18, No. 7).
Issue, p. 609, 1990).

[0004] The immune function of the living body is mainly borne by leukocytes such as granulocytes, monocytes, macrophages, and lymphocytes. In recent years, physiologically active substances such as various cytokines produced by these cells are immunized by the living body. It has been found to be important in maintaining the homeostasis of the mechanism. Even in rheumatic diseases, the relationship between rheumatoid inflammatory properties and cytokines is gradually being clarified. Especially interleukin-
1β (IL-1β) and tumor necrosis factor-α (TNF-
There are many reports on the relationship with α) (Earthris and Rheumatism, Vol. 30, p. 562, 1987). According to these reports, rheumatism is particularly regarded as one of inflammatory diseases, and the relationship between local inflammation and cytokines is discussed. Also, the association with interleukin 6 (IL-6) and granulocyte / monocyte macrophage stimulating factor is becoming clearer (History of Medicine, Vol. 161, 580, 1992). This report also argues, if anything, the relationship as an inflammatory disease, not just rheumatism. Therefore, it is important to examine the function of these leukocytes to produce physiologically active substances such as cytokines in order to determine the pathology of rheumatic diseases.

[0005] As a method for measuring the function of producing a physiologically active substance of leukocytes, there have been reported various methods for examining the function of producing cytokines from blood or leukocytes separated from blood. For example, Japanese Unexamined Patent Publication No. 01-503331, Japanese Unexamined Patent Publication No.
No. 96961 and Japanese Patent Laid-Open No. 03-285692 disclose TNF-production induced by reacting lipopolysaccharide or lectin with blood.
Methods for measuring cytokines such as α and IL-1β are disclosed. Also, JP-A-06-209992, JP-A-07-209992
JP-67955 discloses a method of inducing the production of TNF-α by bringing blood into contact with a polymer material having a specific surface roughness or a polymer material having a specific chemical structure. Also, JP-A-07-299732, JP-A-07-151752
Japanese Patent Application Laid-Open Publication No. H11-139,086 discloses a biological reaction test method in which blood is brought into contact with a polymer material having a specific surface roughness, and the amount of TNF-α and IL-1β production is measured. In addition, methods for testing the effects of various drugs on the above-mentioned cytokine production function have been reported. For example, Japanese Patent Publication No. 7-500905 discloses a method for measuring the activity of an immunomodulator on cytokine production from human peripheral blood leukocytes.

[0006] However, when blood is collected and the amount of cytokine production is measured in a specific reaction container, for example, a blood collection device such as a syringe, a reaction container, or an antigen used as a stimulant is originally used. When the endotoxin (LPS) derived from a Gram-negative bacterium is contaminated, it has been a major problem of the conventional method that accurate measurement becomes difficult. Since endotoxin induces the production of various cytokines from leukocytes in a very small amount, for example, a small amount of endotoxin is contaminated by the contamination of the washing water used in the manufacturing process due to contamination of a small amount of endotoxin by the blood collection device or the reaction vessel. Or when mixed with an antigen used as a stimulant, it is impossible to obtain a reliable measurement result.
For example, "Cytokine production by phytohemagglutini
n stimulated human blood cells: Effect of cortico
steroids, T Cell immunosuppressants and phosphodies
terase4 inhibitors '' J. Van Wauwe, F. Aerts, H. Walter
, and M. de Boer, Inflamm Res 44, 400-405 (1995)
Contains endotoxin mixed with phytohemagglutinin used as a stimulant, IL-2, IL-5, γ-IN
It has been reported that F greatly affects the production of GM-CSF.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for judging the state of a rheumatic disease which can measure the activity of a rheumatic disease with higher accuracy than before, and which is simpler and less dangerous. It is to be.

[0008]

According to the first aspect of the present invention,
Collecting blood in a measurement container, reacting with a material that induces the production of a physiologically active substance in the blood by contacting the blood, and measuring the amount of the physiologically active substance produced in the blood. This is a method for determining the condition of a rheumatic disease. The invention according to claim 2 is characterized in that a material that induces the production of a physiologically active substance in blood when brought into contact with blood is stored in advance in a state where the material can come into contact with blood. This is a method for judging the condition of rheumatic diseases. The invention according to claim 3 is the method for judging a disease state of rheumatic disease according to claim 1 or 2, wherein the inside of the measurement container is evacuated. According to a fourth aspect of the present invention, the material that induces the production of a physiologically active substance in the blood when brought into contact with blood is endotoxin, and the endotoxin content in the measurement container before use is reduced by the amount of the liquid to be measured. The rheumatic disease according to any one of claims 1 to 3, wherein a concentration in the extract when the same water is collected and extracted is 0.5 EU / ml or less. This is a method for determining a disease state. The invention according to claim 5 is
Endotoxin is a material that induces the production of a physiologically active substance in blood when brought into contact with blood, and the concentration of the endotoxin in the whole liquid upon contact with blood is 0.
The method for determining a disease state of a rheumatic disease according to any one of claims 1 to 4, wherein the method is set to be 6 to 100,000 EU / ml. The method according to any one of claims 1 to 5, wherein the amount of the physiologically active substance produced in the blood is determined by enzyme immunoassay. It is.

As mentioned above, the definition of rheumatism is "bone,
A disease that complains of pain and stiffness in motor organs such as joints, muscles, and tendons. " Myositis, dermatomyositis, necrotizing vasculitis and other vascular disorders, diffuse connective tissue disease such as Sjogren's syndrome; arthritis associated with spondylitis such as ankylosing spondylitis, Reiter's syndrome; osteoarthritis .

The materials which induce the production of the above-mentioned physiologically active substances include those which react with blood cells, in particular, leukocytes such as granulocytes, monocytes, macrophages and lymphocytes, promote the activation of these cells, and produce various physiologically active substances. It refers to a material that causes the production, release or induction of a substance, and includes various materials conventionally known as the cell activating substance. For example, various microorganisms such as BCG killed bacteria and genus Corynebacterium, synthetic lipid A, pyran copolymer, purified tuberculin, lectins (phytohemagglutinin, concanavalin A, pork weed mitogen, etc.), OK432 (picibanil), PSK (crestin) , Lentinan, zymosan, LPS (lipopolysaccharide), calcium ionophore, phorbol ester, immunoglobulin-immobilized carrier, formylmethionylleucylphenylalanine (F
Formyl peptides such as MLP) and various cytokines. In particular, endotoxin (LPS) is suitable because of its high activity of inducing a physiologically active substance.

The necessary amount of the above-mentioned material for inducing the production of the physiologically active substance is changed for each material to be used, the inducing activity of the physiologically active substance is measured as an index, and the optimum amount is determined from the dose-response curve. Set. In one embodiment of the present invention, endotoxin (LPS) is used as a material for inducing the production of a bioactive substance, and the bioactive substance used as an index is TNF-α.
In this case, the concentration of endotoxin in whole blood when it comes into contact with blood is preferably 0.6 to 100,000 EU (international endotoxin unit) / ml.

Examples of the physiologically active substances produced in the blood include tumor necrosis factor α (TNF-α), tumor necrosis factor β (TNF-β) IL-1, IL-2, IL-
3, IL-4, IL-5, IL-6, IL-7, IL-
8, IL-9, IL-10, IL-11, IL-12,
Interleukins such as IL-13, IL-14, IL-15, IL-16, INF-α, INF-β, INF-
interferon such as γ, G-CSF, GM-CSF,
Colony stimulating factors such as M-CSF, various cytokines such as RANTES, prostaglandins such as PGE ₂ and PGI ₂ , leukotrienes such as LTB4 and LTC4, nitric oxide, active oxygen, histamine, platelet activating factor (PAF) And various chemical mediators. In addition, an adhesion factor such as soluble ICAM1 and a soluble cytokine receptor such as a soluble IL-2 receptor can be mentioned.

The endotoxin content before use in the measuring container used in the present invention is determined by extracting endotoxin-free water of the same amount as the collected blood volume into the container and extracting the mixture under stirring at 37 ° C. for 1 hour. When performed, it is preferable that the endotoxin content in the extract is 0.5 EU / ml or less. The amount of endotoxin-free water at the time of performing the above extraction is not necessarily required to be exactly equal to the amount of blood collected to be measured as described above. Although it may be less than the amount, even in this case, the endotoxin content in the extract is preferably 0.5 EU / ml or less.

As will be apparent from the experimental examples described below, when the amount of endotoxin in the measuring container exceeds 0.5 EU / ml, cytokine induction is remarkably caused. Therefore, in order to accurately determine the disease state of a rheumatic disease according to the present invention, the endotoxin content in the measurement container before use must be at a level that does not induce cytokine induction.

The method for measuring the endotoxin content in the present invention is a colorimetric method using the color development by hydrolysis of a chromogenic synthetic substrate as an index in the 13th revised edition of the Japanese Pharmacopoeia, "Endotoxin test method". (Manufactured by Seikagaku Corporation)

In the present invention, as a method for removing or inactivating endotoxin, heat treatment, inactivation by acid / alkali treatment, ultrafiltration by a membrane filter, specific treatment for anionic chitosan resin and endotoxin. Various known methods can be used, such as a method of removing an antibody against polymyxin B and endotoxin that binds to a cell with an adsorbent immobilized thereon. In addition, for materials that induce physiologically active substances other than endotoxin and blood anticoagulants, their activity is changed by inactivation by heat treatment or acid / alkali treatment when the level is below the level that does not induce cytokine induction. Fear,
Ultrafiltration and removal with an adsorbent are preferred. Also,
If the device and container used for the endotoxin removal operation are made of glass, they are subjected to a dry heat treatment at 250 ° C. for 1 hour or more. Wash with
Endotoxin must be inactivated.
In addition, it is necessary to use endotoxin-free water, and the operation environment is preferably implemented in an environment that can prevent secondary endotoxin contamination as much as possible, such as a clean room.

In the present invention, when blood is put into the measurement container, secondary endotoxin contamination is caused,
As a result, induction of the physiologically active substance may occur and accurate measurement may not be performed. Therefore, it is necessary to store a material for inducing the production of the physiologically active substance in a measurement container in advance in a state in which the substance can be brought into contact with blood. Is preferred. Further, it is more preferable to reduce the pressure inside the measurement container so that a predetermined amount of blood can be collected.

The shape of the measuring container used in the present invention may be any shape as long as the reaction between the blood and the material for inducing the production of the physiologically active substance can be performed. After the reaction, the blood is transferred to another container. A tube-shaped one such as a blood collection tube or a test tube which can be centrifuged even without it is desirable.

Among the above-mentioned measuring vessels, the tubular one is preferably, for example, a bottomed tubular body having one end opened and the other end closed, and the opening is preferably closed by a stopper. As the bottomed tube, for example, a test tube suitable for centrifugation for measuring the physiologically active substance after the reaction is more preferable, and as the size, the outer diameter is 5 to 30 mm, which is high. It is preferably about 20 to 150 mm.

Examples of the material of the reaction vessel include plastic and glass. As the plastic, either a thermoplastic resin or a thermosetting resin is used. Examples of the thermoplastic resin include polyethylene, polypropylene, polystyrene, polymethyl methacrylate, polyvinyl chloride, polyethylene terephthalate, styrene-acrylonitrile copolymer, and styrene-
Maleic anhydride copolymer, styrene-acrylic acid copolymer, styrene-methyl methacrylate copolymer, ethylene-propylene copolymer, ethylene-acrylic acid copolymer, ethylene-acrylate copolymer, and the like. . Examples of the thermosetting resin include an unsaturated polyester resin, an epoxy resin, and an epoxy-acrylate resin.

In the case where a tubular container is used as the measuring container, and when the inside of the container is maintained at a reduced pressure, it is usually preferable to use a stopper. Examples of the material of the stopper include butyl rubber, chlorinated butyl rubber, and thermoplastic elastomer.

The degree of pressure reduction in the measurement container may be a pressure at which normal-pressure sample blood can be sucked into the container, and the pressure is determined by the amount of sample blood to be sucked. That is, it is necessary to increase the degree of decompression as the amount of the sample blood to be inhaled increases. When a tube-shaped measurement container is used, the amount of blood to be collected varies depending on the volume of the measurement container. However, when a container having a volume of 4 to 5 ml is used, it may be about 0.5 to 2 ml. When a microplate is used, the collected blood volume is 50 μl to 2 μl.
It may be about ml.

The reaction container may contain a blood anticoagulant, if necessary, so as not to coagulate the blood. Examples of the blood anticoagulant include heparin compounds, citrate compounds, oxalate compounds, and the like. Heparin sodium and the like are preferable because they do not inhibit the biological reaction of cells. As the amount of the heparin sodium stored in the container, when blood is stored in the container, if the concentration in the blood is low, blood coagulation may occur, and if the concentration is high, cells may be unexpectedly activated or inactivated. 4 ~
It is stored at 50 u / ml, more preferably 8 to 20 u / ml.

As an example of the method for manufacturing the above-mentioned measuring container, a material capable of inducing the above-mentioned physiologically active substance and a blood anticoagulant are added to a container capable of reducing the pressure inside, and the container is placed in a predetermined reduced pressure state. After that, the container is manufactured by plugging the container. Further, it is preferable that the measurement container is manufactured in a clean environment as much as possible in order to avoid contamination with endotoxin and various bacteria, and if possible, it is preferable to perform a known sterilization treatment after the manufacture.

Next, as one embodiment of the present invention, a measuring container manufactured by adding the above-mentioned material for inducing a physiologically active substance and a blood anticoagulant to the measuring container capable of reducing the pressure inside is used. Next, a method for determining the pathology of a rheumatic disease will be described. First, a blood vessel or a blood collection container is connected to the measurement container using, for example, a multiple injection needle (a blood collection needle whose both ends can be squeezed and communicated), and a sample blood is sucked into the measurement container. Next, while appropriately shaking the measurement container, the blood cells are allowed to react with the material for inducing the physiologically active substance, thereby producing a physiologically active substance such as a cytokine from the blood cells. After the reaction, the cells are separated into blood cells and plasma by standing or centrifugation, and the plasma concentration of a physiologically active substance such as a cytokine produced from the blood cells is determined using a reagent capable of measuring the concentration. As a method of communicating the blood collection container and the measurement container,
For example, there is a method in which a blood collection container is set as a syringe with an injection needle, and the injection needle is pierced into the stopper of the measurement container. The reaction temperature between the blood and the material for inducing the physiologically active substance is 15 ° C. to 4 ° C., which is a temperature at which the production of the physiologically active substance is performed efficiently and does not cause excessive hemolysis.
2 ° C is preferable, and more preferably 30 ° C to 40 ° C. The reaction time is a reaction time at which the production of the physiologically active substance is performed efficiently and does not cause excessive hemolysis.
The time is preferably from 48 to 48 hours, more preferably from 2 to 24 hours.

As a reagent for measuring a physiologically active substance produced from blood cells, which is used in the method for determining a pathological condition of a rheumatic disease of the present invention, any reagent which can quantify the amount of the above-mentioned physiologically active substance is used. Although not particularly limited, for example,
Examples include a monoclonal or polyclonal antibody against a physiologically active substance to be quantified, an enzyme immunoassay reagent utilizing enzymes such as peroxidase and alkaline phosphatase, and a coloring substrate of each enzyme.

Hereinafter, one embodiment of the method of the present invention for quantifying the plasma concentration of a physiologically active substance such as cytokines produced from blood cells by enzyme immunoassay will be described. First, blood is reacted with a material for inducing the physiologically active substance in the measurement container to induce a physiologically active substance, and the blood after induction is centrifuged at 1600 G to separate a blood cell component and a plasma component. Next, the separated plasma is added by pipetting to the wells of a microplate on which the monoclonal antibody against the physiologically active substance has been immobilized, and reacted at 37 ° C. for 2 hours. Subsequently, the plasma solution after the reaction is discarded by a means such as aspiration and the like, and further, in order to remove unreacted components, the wells are washed with a neutral pH washing buffer containing a nonionic surfactant such as Tween 20. Wash. Next, a polyclonal antibody against the above-mentioned physiologically active substance on which horseradish peroxidase is immobilized is added by pipetting, followed by reaction at 37 ° C. for 1 hour. Next, in order to remove unreacted horseradish peroxidase-immobilized antibody, the well is washed with the above-mentioned washing buffer, and a substrate solution containing hydrogen peroxide and tetramethylbenzidine is added, followed by reaction for 5 to 10 minutes. Next, 1 M sulfuric acid is added to stop the reaction, and the color of the substrate due to the enzyme reaction is measured from the absorbance at 450 nm. From the measured value and a calibration curve prepared using a known concentration of the bioactive substance, the amount of the bioactive substance production induction is measured.

[0028]

The present invention will be described in more detail below by giving Examples and Comparative Examples of the present invention. The present invention is not limited to the following examples. Glassware,
The container is subjected to a dry heat treatment at 250 ° C. for 2 hours, and a plastic device and a commercially available endotoxin-free one are used, or the endotoxin is inactivated by previously immersing in a 0.2 M aqueous sodium hydroxide solution overnight. Washed thoroughly with endotoxin-free water was used. The operation was performed in a clean bench.

Example 1 A 5 ml blood collection tube (12.6φ × 75 mm) made of endotoxin-free (endotoxin content in an extract is less than 0.5 EU / ml) polyethylene terephthalate as a measurement container before use (12.6 φ × 75 mm) (Manufactured by Sharp Corporation). E. coli 055B5-derived endotoxin (manufactured by LBL) was added to this measuring container in 100 EU portions, and dried under reduced pressure. Next, a butyl rubber stopper that fits the diameter of the tube is lightly placed on the opening of the test tube so as not to seal the opening, and then placed in a container that can be decompressed so that about 0.5 ml of blood can be sucked. Then, when the pressure was reduced to 665 mmHg, the opening of the test tube was sealed. MRL / Mp-lpr / lpr mouse (hereinafter abbreviated as MRL / lpr mouse, 16 weeks old, male) 3
Two were purchased from SLC Japan and bred in a clean environment. Eight animals were randomly selected at 17 weeks, 21 weeks, 25 weeks, and 29 weeks of age, and heparin sodium (Novo-Heparin Injection, manufactured by Novo Nordisk A / S) was selected from each mouse. 1000) A syringe pre-stored with 20 μl was provided with a 27G injection needle, and heart blood was collected in approximately 1.5 ml portions. The needle of the syringe from which the blood was collected was pierced into the stopper of the measurement container to which the endotoxin was added, and about 0.5 ml of blood was added to each measurement container. Then, the reaction was carried out at 37 ° C. for 4 hours while slowly stirring. After completion of the reaction, the mixture was centrifuged at 1600 G for 10 minutes at 4 ° C., and the supernatant plasma was collected.

The amount of TNF-α in the collected plasma was determined as TNF-α.
The measurement was performed using an enzyme immunoassay kit using an α monoclonal antibody. The amount of TNF-α production was measured using Factor-Test-X ^™ Mouse TNF-α ELISA KIT (trade name, manufactured by Genzyme). Note that the measurement limit concentration of this measurement method is 15 pg /
ml. In order to determine the endotoxin content in the measurement container before use, the same Lo as the measurement container used was used.
The measurement was performed as follows using the blood collection tube of t. About 1 ml of endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd.) was collected in the above measuring container, and stirred at 37 ° C. for 1 hour to extract endotoxin. Next, the endotoxin content in this extract was measured using the Endospecy ES50M set (manufactured by Seikagaku Corporation).
Was measured by a microplate method and a red measurement method. As a result, in all of the test containers tested, the 0.
The amount of endotoxin was lower than 5 EU / ml. The severity of spontaneous rheumatoid polyarthritis in each MRL / lpr mouse was comprehensively judged from the degree of deformity of the limb finger joints and the degree of tail joint deformity. Classified. 0: Apparently unchanged from normal mice. +1: The tail is slightly deformed. +2: The tail is considerably deformed. +3: The claws of the limbs are peeled off, and there is bleeding. Table 1 and FIG. 1 show the scores obtained by classifying spontaneous rheumatoid polyarthritis of each MRL / lpr mouse from the above scale and the TNF-α production ability when LPS was used as a stimulating material.

[0031]

[Table 1]

Comparative Example 1 Each MR collected in Example 1
Serum was collected from the blood of L / lpr mice, and R
The F amount was measured using a reagent for RF measurement (Auto TIA RFII “Nissui”, manufactured by Nissui Pharmaceutical Co., Ltd.). The measurement results are shown in Table 1 and FIG. As is clear from Table 1 and FIGS. 1 and 2, the transition of the severity of the symptoms of spontaneous rheumatoid polyarthritis in MRL / lpr mice and the TNF-α production ability when LPS was used as a stimulating material were examined. The transition showed good agreement. In contrast, measurements of RF in serum did not always match the severity of the symptoms.

Example 2 MRL / lpr mice (16 weeks old,
(Male) 8 animals were purchased from SLC Japan and bred in a clean environment. 4 each at 17 and 29 weeks of age
20 μl of sodium heparin (Novo Nordisk A / S, trade name Novo Heparin Note 1000) from one mouse
A syringe with 27 g was previously fitted with a 27G injection needle, about 1.5 ml of blood was collected from the heart, and pooled in a syringe for 10. A 22G injection needle was attached to the syringe containing the pooled blood, and 1 endotoxin was administered in the same manner as in Example 1.
00EU was added, and 0.5 ml of blood was pierced under reduced pressure into the stopper of a tightly sealed measurement container so that 0.5 ml of blood could be collected, and about 0.5 ml of blood was added to each of the five reaction containers. Then 3
The reaction was carried out at 7 ° C. for 4 hours with slow stirring. After the reaction is completed, centrifuge at 1600 G for 10 minutes at 4 ° C.
The supernatant plasma was collected. Then, TNF-α in plasma
The amount was measured in the same manner as in Example 1, and the simultaneous reproducibility was evaluated.

(Comparative Example 2) A 5 ml blood collection tube (12.6φ × 75 mm) made of polyethylene terephthalate without endotoxin free (endotoxin content in the extract is less than 0.5 EU / ml) as a measurement container before use
The test was performed in the same manner as in Example 2 except that Sekisui Chemical Co., Ltd. was used. The same blood used in Example 2 was used as the blood used, and the simultaneous reproducibility in five measurement containers was evaluated.
In addition, in order to determine the endotoxin content in the measurement container before use, the measurement was performed as follows using a blood collection tube of the same Lot as the measurement container used. About 1 ml of endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd.) is collected in the measurement container,
After stirring for 1 hour at 37 ° C., endotoxin was extracted.
Next, the endotoxin content in this extract was measured by a microplate method and a red measuring method using Endospecy ES50M set (manufactured by Seikagaku Corporation). as a result,
Endotoxin higher than 0.5 EU / ml was measured in all of the test containers tested.

Table 2 shows the results of Example 2 and Comparative Example 2. As is clear from the results, the endotoxin content in the measurement container before use was 0.5% as the concentration in the extract.
When a container higher than EU / ml is used, simultaneous reproducibility is poor in the blood of MRL / lpr mice of either 17 weeks or 29 weeks of age, and the endotoxin content in the measurement container before use is low. 0.5 EU / ml as the concentration in the extract
Good simultaneous reproducibility was shown when less than 10 containers were used. In addition, when a container having an endotoxin content higher than 0.5 EU / ml as the concentration in the extract was used, the measurement container was originally contaminated, so that even a 17-week-old mouse had a high level of TNF-α. Production is seen,
There were no significant differences from the 29 week old mice, and did not reflect the severity of arthritis symptoms. On the other hand, when a container having an endotoxin content of less than 0.5 EU / ml as the concentration in the extract was used, a significant difference was observed depending on the severity of arthritis symptoms.

[0036]

[Table 2]

(Reference Example 1) Endotoxin-free (0.5 EU as a concentration in an extract) was used as a measurement container before use.
5 ml of polyethylene terephthalate)
(12.6φ × 75 mm) (manufactured by Sekisui Chemical Co., Ltd.). Syringe (Termo syringe manufactured by Terumo)
In advance, heparin sodium (Novo Nordisk A / S, trade name: Novo Heparin Note 1000) was added in advance of 35 u.
Then, 20 ml of blood was collected from the elbow vein of a healthy person. About 1 ml of this blood was dispensed into the measuring container before use, and E. coli UKT-B-derived endotoxin (Japanese Pharmacopoeia standard) was added to each measuring container at 0 EU / ml and 0.1 EU / m.
l, 0.2EU / ml, 0.5EU / ml, 1EU / ml, 2EU / ml
0.05 EU / ml and 0.05 EU / ml were added. Then, the reaction was carried out at 37 ° C. for 4 hours while slowly stirring. After completion of the reaction, the mixture was centrifuged at 1600 G for 10 minutes at 4 ° C., and the supernatant plasma was collected. The amount of each cytokine of TNF-α, IL-1β, and IL-6 in the collected plasma was measured using an enzyme immunoassay kit using each monoclonal antibody. Measurement of the amount of TNF-α induction
Genzyme brand name, PREDICTA Human TNF-α ELISA K
Performed using IT. In addition, the detection limit concentration of this measurement method was 15 pg / ml. In addition, measurement of the amount of IL-1β induction was carried out using a product name of Genzyme, PREDICTA Human IL-1β E
Performed using LISA KIT. In addition, the detection limit concentration of this measurement method was 35 pg / ml. Moreover, the measurement of the amount of IL-6 production was performed using PREDICTA Human IL-6 ELISA KIT. The detection limit concentration of this measurement method was 35 pg / m.
l. The results are shown in Table 3. As is clear from these results, when the endotoxin content is greater than 0.5 EU / ml in the extract, TNF-α, IL
It is clear that induction of production of physiologically active substances such as -1β and IL-6 occurs.

[0038]

[Table 3]

[0039]

According to the present invention, since the measurement container in which the endotoxin content is controlled is used, there is no problem that unnecessary activation of the cells is caused, and the physiologically active substance such as cytokines of blood cells, especially leukocytes, is accurately obtained. Production capacity can be measured. Also, whole blood can be used as it is,
Since there is no need to separate monocytes and lymphocytes from blood, the operation is simplified, and the problem of reduced activity accompanying the separation operation is also eliminated. Also, after collecting blood from the subject, transfer the blood to various reaction vessels by means such as pipetting,
Since no operations such as cell separation and cell culture are required, the tester has little risk of being infected with various infectious diseases such as hepatitis and AIDS. In addition, special techniques such as cell separation, cell culture, and microscopic measurement are not required, the measurement time can be reduced, and expensive equipment such as an RI facility or a flow cytometer is not required. The present invention enables safe, accurate, and simple determination of the state of a rheumatic disease by using the ability to produce an active substance as an index.

[Brief description of the drawings]

FIG. 1 shows the severity of arthritis and TNF- in Example 1.
Diagram showing the relationship with α production

FIG. 2 is a diagram showing the relationship between the severity of arthritis and the amount of RF in Comparative Example 1.

Claims (6)

[Claims] 1. Blood is collected in a measuring container, and is brought into contact with blood to react with a material that induces the production of a physiologically active substance in the blood, and the amount of the physiologically active substance produced in the blood is measured. A method for determining the condition of a rheumatic disease. 2. A material for inducing the production of a physiologically active substance in blood by being brought into contact with blood, the material being stored in advance in a state capable of contacting with blood.
The method for determining a disease state of a rheumatic disease according to the above. 3. The method for judging a disease state of a rheumatic disease according to claim 1, wherein the pressure in the measurement container is reduced. 4. A material which induces the production of a physiologically active substance in blood when brought into contact with blood is endotoxin, and the endotoxin content in a measurement container before use is equal to the amount of liquid to be measured. The method for judging a disease state of a rheumatic disease according to any one of claims 1 to 3, wherein the concentration in the extract at the time of collection and extraction is 0.5 EU / ml or less. . 5. A material which induces the production of a physiologically active substance in blood when brought into contact with blood is endotoxin, and when the endotoxin is brought into contact with blood, the concentration of the endotoxin in the whole liquid is from 0.6 to 0.6. The method for determining a disease state of a rheumatic disease according to any one of claims 1 to 4, wherein the method is adjusted to 100,000 EU / ml. 6. The method according to claim 1, wherein the amount of the physiologically active substance produced in the blood is quantified by enzyme immunoassay.
The method for judging the condition of a rheumatic disease according to any one of claims 1 to 5.