JP2001194366A - Container, kit and method for measuring cell function - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a container, a kit and a method for measuring the cell function which are simple in operation, free from any dangers, and capable of measuring the cell function with high accuracy. SOLUTION: The endotoxin of the quantity sufficient for producing the physiologic active substance in the blood cell is stored in a contactable manner with the sampled blood in the container for measuring the cell function which is used in measuring the physiologic active substance produced by the blood cell in which the quantity of the endotoxin in the container is specified not to affect the measured value of the physiologic active substance, and the concentration in the total solution in contact with the blood of the endotoxin is set to be 0.6-1000 EU/ml.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell function measuring container, a cell function measuring kit and a cell function measuring method for measuring cell functions involved in an immune response, an inflammatory response and the like. The present invention relates to a cell function measurement container, a cell function measurement kit, and a cell function measurement method for measuring a cell function by measuring a physiologically active substance such as a cytokine produced by blood cells.

[0002]

2. Description of the Related Art Leukocytes such as granulocytes, monocytes, macrophages, and lymphocytes play various roles in various biological defense reactions such as an inflammatory response and an immune response in blood and in each organ or organ. These cells play an important role in various pathological conditions such as inflammatory diseases such as infectious diseases and hepatitis; immunological and allergic diseases such as rheumatoid arthritis and asthma; and cancers. Is known to be suppressed or enhanced.

[0003] In addition, anti-inflammatory drugs,
Various drugs such as immunosuppressants, immunopotentiators, and anticancer drugs have been used, and it is also known that the function of these cells is suppressed or enhanced in such cases. Therefore, it is important to examine the functions of these cells in order to grasp the pathological condition of various diseases, the effects or side effects of drugs, determine treatment guidelines, and determine the dose and timing of drugs.

Conventionally, in an examination room or an examination center of a hospital,
For the reasons described above, a granulocyte phagocytic function test, a granulocyte bactericidal ability (active oxygen-producing ability) test, a lymphocyte blastogenesis test and the like have been performed to measure the cell function. Recently, surface antigen tests using a flow cytometer and a fluorescently labeled monoclonal antibody against various immunocompetent cell surface antigens have been performed.

However, the conventional test methods require special techniques such as cell separation, cell culture, and microscopic measurement.
The measurement required a long time, and required expensive equipment such as an RI facility and a flow cytometer. In addition, these cell function measurement methods mainly target granulocytes and lymphocytes, and do not examine the functions of monocytes and macrophages.

[0006] Monocytes in blood and macrophages which have been differentiated and matured from monocytes in tissues are involved in elimination of foreign substances through phagocytosis, establishment of immunity through antigen presentation, and cytokines and prostasis. By secreting various physiologically active substances such as glandin, it has a very diverse function, such as regulating inflammatory and immune responses. Therefore, monocytes and macrophages play an important role in various pathological conditions as well as granulocytes and lymphocytes, and it is very important to examine the functions of these cells.

[0007] In particular, in infectious diseases and the like, monocytes and macrophages differ from granulocytes and lymphocytes in that the number of cells does not fluctuate so much, their functions are mainly amplified, and it is more important to measure changes in cell functions. ("Macrophage" Toru Tokunaga, Kodansha Scientific 1986 first edition).

[0008] Tumor necrosis factor α (hereinafter referred to as TNF-α), interleukin-1β (hereinafter referred to as IL-1β), interleukin-6 (hereinafter referred to as IL-6)
Is called a monokine, which is mainly produced by monocytes and macrophages in blood cells and is a cytokine involved in various inflammatory and immune responses.

Hitherto, various methods have been reported for examining the above-mentioned cytokine production function from blood or leukocytes separated from blood. For example, Japanese Translation of International Patent Publication No. 1-503331
JP, JP-A-2-196951, JP-A-3-2
No. 85692 discloses that TNF-α and IL-induced production of lipopolysaccharide (LPS) or lectin by reacting with blood.
A method for measuring a cytokine such as 1β is disclosed.

Japanese Patent Application Laid-Open Nos. Hei 6-209998 and Hei 7-67955 disclose a method in which blood is brought into contact with a polymer material having a specific surface roughness or a polymer material having a specific chemical structure. Methods for inducing -α production have been disclosed.

JP-A-7-299732, JP-A-7-29932
Japanese Patent Publication No. -151752 discloses a biological reaction test method in which a polymer material having a specific surface roughness is brought into contact with blood to measure the amount of TNF-α or IL-1β produced.

Japanese Patent Publication No. 7-500905 discloses a TN
A method for measuring the immunoreactive ability of a test substance by measuring the amount of F-α or IL-1β induced to produce cytokines from human peripheral blood leukocytes is disclosed.

[0013] However, the following problems have been encountered in the cell function measuring method conventionally performed in the examination room or the examination center of the hospital and the method disclosed in the above-mentioned patent publication.

That is, in all of these tests, after blood is collected from a subject using a syringe, blood is transferred to various reaction vessels by a technique such as pipetting, and cell separation and function for separating leukocytes and the like are performed. Special operations such as cell culture for measurement were required. Therefore, there is a risk that the test worker may come into contact with various infectious diseases such as hepatitis and AIDS by touching the blood.

Further, during these operations, various sterilization and dust may be mixed during the blood test, and the cells in the blood are unnecessarily stimulated by these contaminants or physical stimulation by the operations. In addition, there is a possibility that the measurement results may be adversely affected.

Further, blood is collected, and in a specific reaction vessel, cytokines from leukocytes, in particular, TNF-
As another problem of the conventional method for measuring the production amounts of α, IL-1β and IL-6, for example, endotoxin such as LPS originally derived from Gram-negative bacteria is mixed in a blood sampling device such as a syringe or a reaction vessel. If you do. Endotoxin is minimal in leukocytes from TNF-α, IL
In order to induce the production of -1β and IL-6, for example, when a small amount of endotoxin is mixed into the blood collection device or the reaction container due to contamination of a small amount of dust in a manufacturing process or contamination from a washing solution to be used, it is difficult to be reliable. It was not possible to obtain a possible measurement result.

Because of the above-mentioned problems, there is a demand for a method for measuring cell functions which is easier and less dangerous than the conventional methods and has a high accuracy. On the other hand, blood anticoagulants have conventionally been used in the measurement of various physiologically active substances in blood, the function of blood cells, and the measurement of surface antigens on blood cells. However, there is no general standard for the endotoxin content of anticoagulants, and for anticoagulants used as injections, the 13th revised edition of the Japanese Pharmacopoeia, "Endotoxin Test Method," There is only a guideline that uses 5 EU / kg as the standard for setting the standard value. Endotoxin is a lipopolysaccharide which is a cell wall outer membrane component of Gram-negative bacteria, and stimulates TNF-
It produces bioactive substances such as various cytokines such as α, IL-1β, IL-6 or granulocyte-macrophage colony stimulating factor, and has various physiological actions such as pyrogenic activity and endotoxin shock (Nippon Medical Center) "Inflammation and Cytokine '87 Inflammation Seminar" p103-10
8). In addition, physiologically active substances such as the above cytokines produced from blood cells act on autocrine and paracrine, causing further production of histamine or arachidonic acid metabolites or various cytokines, and various functions of blood cells. Or cause quantitative and qualitative changes in blood cell surface antigens. Therefore, if the blood anticoagulant is contaminated with the endotoxin content, and the endotoxin content is at a level that causes the production of a bioactive substance, the measurement of various bioactive substances in blood, the measurement of blood cell function, and the measurement of blood cells It is impossible to accurately measure the surface antigen.

[0018]

In view of the above points, the present inventors have disclosed in WO98 / 07031, human peripheral blood leukocytes as 0.6-100,000 EU.
The present invention discloses a method of measuring cytokines such as TNF-α and IL-1β induced by stimulating with an endotoxin controlled in the range of / ml, eliminating the disadvantages of the prior art,
A cell function measurement container, a cell function measurement kit, and a cell function measurement method that are easy to operate, have no danger, and can measure cell functions with high accuracy have been disclosed. However, when human peripheral blood leukocytes are stimulated with a low-concentration endotoxin of 0.6 to 1000 EU / ml, the amount of cytokine produced is almost plateau (plateau). , The amount of cytokine produced changes rapidly. On the other hand, when accommodating endotoxin in a cell function measurement container, it is very difficult to control the content thereof with extremely high precision. From the above,
When stimulating with the above-mentioned high-concentration endotoxin, there is a problem that a slight error in the endotoxin content causes a large variation in the measured value and deteriorates the measurement accuracy.

In a conventional study, a high concentration of endotoxin (10,000 to 100,000 EU / ml) has been used as a stimulant. However, high endotoxin stimulation was a test in non-physiological conditions.
In fact, it has been reported that the concentration of endotoxin in the blood of patients with Gram-negative bacterial infection is at most 0.5 to 5 EU / ml. Evaluation, basic and clinical
p173-179, Vol. 26, No. 11, September 20, 1992). Under such conditions, various cells may be stimulated non-specifically. Usually, it is considered that endotoxin-stimulated production of cytokines such as TNF-α occurs from monocytes and macrophages. However, it has been reported that TNF-α is also produced from granulocytes when stimulated with high concentration of endotoxin (Dominik B. Dubravec: “Circulating huma”).
n peripheral blood granulocyte synthesize and secr
ete tumor necrosis factor α ", Proc. Natl. Acad. S
ci. USA, Vol. 87, pp6758-6761, 1990), and a measurement using a conventional high-concentration endotoxin could not obtain a result specific to monocytes and macrophages.

The present invention has been made in view of the above problems, and has as its object to solve the drawbacks of the conventional method, to measure the cell function more easily, without danger and with higher accuracy than the conventional method. It is an object of the present invention to provide a cell function measurement container, a cell function measurement kit, and a cell function measurement method that can be performed. In particular, an object of the present invention is to provide a cell function measurement kit that can specifically measure the functions of monocytes and macrophages in blood cells.

[0021]

The present invention according to claim 1 (hereinafter referred to as “the present invention 1”) is a cell function measuring container used for measuring a physiologically active substance produced by blood cells, In a container defined in advance such that the amount of endotoxin in the container does not affect the measured value of the physiologically active substance, a sufficient amount of endotoxin to cause the blood cells to produce the physiologically active substance, and the collected blood and A cell function measurement container housed in a contactable state, wherein the concentration of the endotoxin in the whole liquid when contacted with blood is 0.6 to 1000 EU / ml. I will provide a.

The present invention according to claim 2 (hereinafter referred to as “invention 2”) is a cell function measuring container used for measuring a physiologically active substance produced by blood cells,
A container in which the amount of endotoxin in the container is previously equal to or less than 0.5 EU / ml as the concentration in the extract when water is collected in the container and extracted. A cell characterized in that endotoxin is stored in a state in which it can be brought into contact with the collected blood so that the concentration in the whole solution when it comes into contact with blood becomes 0.6 to 1000 EU / ml. Provide a container for measuring functions.

According to the present invention described in claim 3 (hereinafter referred to as the present invention 3), there is provided a container for measuring cell function according to claim 1 or 2, wherein the container is open at one end and closed at the other end. A cell function measurement container, comprising a bottomed tubular body, and a stopper that is hermetically sealed without being removed at the time of collecting a blood sample, is attached to an opening of the container.

According to a fourth aspect of the present invention (hereinafter referred to as the fourth aspect), there is provided the cell function measuring container according to the third aspect, wherein the pressure in the container is reduced. A measuring container is provided.

In the present invention according to claim 5 (hereinafter referred to as the present invention 5), the cell function measuring container according to any one of claims 1 to 4, and the amount of the induced physiologically active substance. And a reagent for measuring cell function.

In the present invention according to claim 6 (hereinafter referred to as present invention 6), the cell function measuring container according to claim 2 and the amount of endotoxin in the container are equal to the amount of the liquid to be measured. A control value measuring container having a concentration of 0.5 EU / ml or less as a concentration in the extract when water is collected in the container and extracted, and a quantification reagent for quantifying a physiologically active substance. The present invention provides a kit for measuring cell function, comprising:

According to the present invention described in claim 7 (hereinafter referred to as the present invention 7), the cell function measuring kit according to claim 5 or 6 is used to measure the cell function of monocytes and macrophages. Provide a way.

Hereinafter, the present invention will be described in detail. [Inventions 1 to 4 (hereinafter, also simply referred to as the present invention): Cell Function Measuring Container] The cell function measuring container in the present invention comprises:
Prior to storing a sufficient amount of endotoxin in a container to produce a bioactive substance, it is specified that the amount of endotoxin in the container does not affect the measured value of the bioactive substance. Then, in a container in which the amount of endotocin is defined in this manner, a sufficient amount of endotoxin for producing a physiologically active substance is stored. The amount of the stored endotoxin is such that the concentration in the whole liquid when it is brought into contact with blood at the time of collecting a blood sample is 0.6 to 1000 EU.
/ Ml.

As the physiologically active substance in the present invention, for example, TNF-α; IL-1, IL-4, IL-5, I
Interleukins such as L-6; IFNα, IFNβ, I
Interferon such as FNγ; colony stimulating factor; IL
-8, various cytokines such as chemotactic factors such as RANTES, and prostaglandins such as PGE2 and PGI2; leukotrienes such as LTB4 and LTC4; nitric oxide; active oxygen; histamine; platelet activating factor (PA
And various chemical mediators such as F). Adhesion factors such as soluble ICAM1 and soluble I
Also included are soluble cytokine receptors such as L-2 receptor, intracellular granule enzymes such as matrix metalloproteinase and macrophage-specific elastase. Of the above physiologically active substances, more preferably, cytokines are used.

As a method for measuring the above-mentioned physiologically active substance, for example, a monoclonal antibody or a polyclonal antibody against the above-mentioned physiologically active substance to be measured, enzymes such as peroxidase and alkaline phosphatase, and a chromogenic substrate of each enzyme are used. An enzyme immunoassay may be used.

The endotoxin in the present invention includes
Reacts with blood cells, especially granulocytes, monocytes, macrophages, leukocytes such as lymphocytes, and promotes the activation of these cells,
It is a material that induces the above-mentioned physiologically active substances (cytokines and the like), and various materials conventionally known as the above-mentioned cell activators are used. For example, microorganism-derived cell wall polysaccharide (LPS) and various natural substances Alternatively, a material immobilized on a synthetic polymer material or the like is used.

In the container for measuring cell function of the present invention, a container defined so that the amount of endotoxin in the container does not affect the measured value of a physiologically active substance before a sufficient amount of endotoxin is stored. Is determined that the amount of endotoxin in a container is equal to or less than 0.5 EU / ml as the concentration in an extract when water equal to the amount of the liquid to be measured is collected in the container and extracted. Preferably it is a container.

As a specific method for extracting the amount of endotoxin, the same amount of endotoxin-free water as the amount of collected blood is collected in the container, and extraction is performed under stirring at 37 ° C. for 1 hour. The amount of endotoxin-free water at the time of performing the above extraction does not necessarily need to be exactly equal to the amount of blood to be collected as described above, and if the extraction is sufficient, the amount of water to be measured Although it may be less than the blood volume, even in this case, the endotoxin content in the extract is preferably 0.5 EU / ml or less.

The method for measuring the amount of endotoxin 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).

The amount of endotoxin stored in a container in which the amount of endotoxin is predetermined and sufficient for causing blood cells to produce a physiologically active substance is such that the concentration of endotoxin in the whole liquid after blood sample collection is 0.1. 6-1000E
U (international endotoxin unit) / ml, more preferably 8-800 EU / ml
The amount must be The above concentration is 0.6 EU / m
When the amount is less than 1, the amount of induction of TNF-α, IL-1β and IL-6 may be too small, and may be 1000 EU / m 2.
When the value exceeds 1, not only the functions of monocytes and macrophages in blood cells cannot be specifically measured, but also the measurement error increases. The above-mentioned total solution indicates the sum of the solution of blood and endotoxin, and the sum of the solution when a blood anticoagulant is used.

The state of the endotoxin present in the measurement container is usually such that the endotoxin in the form of a powder or a solution dissolved in a solvent such as water is converted into a solid, gel or liquid form in the container. May be present, and
After being dissolved in an appropriate solvent and applied or added to the inner wall surface of the container, it may be present in powder form.

Examples of the solvent include a buffer such as a phosphate buffer and a Hanks buffer, MEM and RPMI-1.
Any physiological buffer such as a normal medium such as 640 can be used. Furthermore, commercially available water for injection (for example, LP
S-free water; Otsuka Pharmaceutical Co., Ltd.) and physiological saline (for example, Otsuka Pharmaceutical Co., Ltd.) can also be used.

As a method for storing the endotoxin,
There is no particular limitation as long as the endotoxin can be brought into contact with blood in the measurement container.

The shape of the cell function measuring container in the present invention is not particularly limited, and examples thereof include a tube shape and a plate shape. As the tube shape, a blood collection tube, a test tube and the like are used. Examples thereof include a microplate and the like, and those capable of performing a decompression operation are preferable.

In the cell function measuring container of the present invention, the pressure in the container is preferably reduced. The degree of the pressure reduction is determined when the blood at normal pressure and the measuring container are connected to each other. The pressure may be such that normal-pressure blood can be inhaled, and the pressure may be determined according to the amount of blood to be inhaled. That is, the greater the amount of blood to be inhaled, the greater the degree of decompression.

Examples of the material of the measuring container 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, styrene-maleic anhydride copolymer, and styrene-acrylic acid copolymer. Polymer, styrene-methyl methacrylate copolymer,
Examples thereof include an ethylene-propylene copolymer, an ethylene-acrylic acid copolymer, and an ethylene-acrylic acid ester copolymer.

Examples of the thermosetting resin include unsaturated polyester resins, epoxy resins, epoxy-acrylate resins and the like.

In the tube of the measuring container, a plug is usually used to maintain the inside of the measuring container at a reduced pressure. Examples of the material of the plug include butyl rubber, chlorinated butyl rubber, and thermoplastic elastomer. The degree of the pressure reduction may be a pressure at which the normal-pressure sample blood can be sucked into the measurement container, and is determined by the amount of the sample blood to be sucked.

In the case of using a tube-shaped measuring container, the amount of blood to be collected varies depending on the volume of the measuring container. It may be about 2 ml. In addition, when using a microplate-shaped thing, the collected blood volume is
It may be about 0.05 ml to 2 ml.

The amount of blood used for measuring the cell function using the cell function measuring container according to the present invention varies depending on the volume of the measuring container. It may be about 0.5 to 2 ml.

As an example of a method for manufacturing the measurement container, the above-mentioned endotoxin and blood anticoagulant are added to a container capable of reducing the pressure inside, and the container is brought into a predetermined reduced pressure state. There is a method of plugging.

As the above-mentioned container, for example, a tube with a bottom having one end opened and the other end closed is preferable, and an opening that can be closed by a stopper is preferable. Further, it is preferable that the stopper is hermetically sealed without being removed at the time of blood sample collection at the opening of the container. Further, as the bottomed tube, for example, a test tube suitable for centrifugation operation for measuring the amount of the cytokine after the induction reaction of the cytokine is more preferable, and as the dimensions thereof,
Those having an outer diameter of about 5 to 30 mm and a height of about 20 to 150 mm are preferred.

The measuring container of the present invention is preferably manufactured in a clean environment as much as possible in order to avoid contamination with endotoxin and various bacteria. preferable.

A method for measuring the cell function of blood cells using the cell function measuring container according to the present invention will be described.
First, a blood vessel or a blood collection container is communicated with the measurement container, and a sample blood is sucked into the measurement container. Next, while appropriately shaking the measurement container, blood cells and endotoxin are brought into contact with each other to cause a reaction to be induced. After the reaction, the cells are separated into blood cells and plasma by standing or centrifugation, and the amount of cytokines in the plasma is quantified with each quantifiable reagent.

The lower the reaction temperature between blood and endotoxin in the measuring container, the lower the metabolic activity of the cells,
The amount of cytokine induction may be too low, and if it is too high, cells may be damaged, and the amount of cytokine induction may be too low.Therefore, since the amount of cytokine induction is low, 26-45 ° C. is preferable, and more preferably , 30-42 ° C.

As is clear from the examples described below, at low concentrations of endotoxin (for example, 100 EU / ml) in the range of 0.6 to 1000 EU / ml as defined in the present invention, the production of TNF-α is One hour after the start of the reaction, the plateau was detected after 4 to 8 hours, and the production amount was reduced after 24 hours. On the other hand, a high concentration of endotoxin (for example, 50,000 EU / ml) which is a conventional method
Upon stimulation, TNF-α production increased up to 8 hours later. That is, 0.6 to 1000E as in the present invention.
The method of stimulating endotoxin at a low concentration of about U / ml can obtain a stable result in a shorter time than the conventional method using a high concentration of endotoxin. From these results, the reaction time between blood and endotoxin in the measurement container is such that, if the reaction time is short, the amount of cytokine induction may be too small, and if the reaction time is too long, the measurement result will be output slowly. Since the amount of induction also tends to gradually decrease with a peak at about 4 hours, it is preferably 1 to 6 hours, more preferably 2 to 4 hours.
Time.

As a method for measuring the cytokine induced by the above method, various known methods are used.
A method using an enzyme immunoreagent is exemplified.

The above enzyme immunoassay reagent is composed of a monoclonal antibody or polyclonal antibody against the above cytokine to be measured, enzymes such as peroxidase and alkaline phosphatase, and a chromogenic substrate of each enzyme. A sandwich enzyme immunoassay in which a monoclonal antibody against the cytokine to be measured is immobilized on the phase surface in advance is preferable because it does not need to be immobilized before measurement and has excellent reproducibility.

Hereinafter, an embodiment of measuring the cell function using the cell function measuring container of the present invention will be described in detail. First, blood and endotoxin are allowed to react in the above-described measurement container to induce cytokines.
Centrifuge at 00G to separate blood cell components and plasma components. Next, the separated plasma and a polyclonal antibody against the cytokine are added to the wells of a microplate by pipetting, and reacted at 37 ° C. for 2 hours. Next, the plasma solution after the reaction is discarded by means such as suction removal, and Tween 2 is used to remove unreacted components.
The wells are washed with a neutral pH washing buffer containing a nonionic surfactant such as 0. Next, a polyclonal antibody against the above-mentioned cytokine immobilized with horseradish peroxidase is added to the above-mentioned wells by pipetting, and the mixture is reacted 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, a 1 M sulfuric acid solution 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 values and a calibration curve prepared using a known concentration of the cytokine, the amount of the cytokine induced is measured.

The measurement container of the present invention may contain a blood anticoagulant, if necessary. Examples of the blood anticoagulant include a heparin compound, a citrate compound, an oxalate compound, and the like. Heparin sodium and the like are preferable because they do not inhibit the biological reaction of cells.

It is preferable that the blood anticoagulant has an endotoxin content in the blood anticoagulant in an amount that does not produce a physiologically active substance from blood cells when mixed with blood. That is, when the endotoxin content of the blood anticoagulant itself is reduced as described above, it is possible to suppress the application of unnecessary stimulus to the collected blood until the test is performed. As a result, the physiologically active substance of the collected blood is
It is difficult to be produced by a blood anticoagulant, and can measure various physiologically active substances and cell functions more accurately. Further, after blood collection, the blood sample can be stored for a long time until the test is performed. The endotoxin content in the blood anticoagulant increases as TNF-α, I
Production of cytokines such as L-1 and IL-6 is caused, and accurate measurement cannot be performed. Therefore, it is desirable to limit the endotoxin content of the blood anticoagulant to an amount that does not produce cytokines as physiologically active substances in the collected blood. When the endotoxin content in the blood anticoagulant exceeds 0.5 EU / ml in the reaction blood, TNF
-Production of cytokines such as α, IL-1, and IL-6 is caused. Therefore, the endotoxin content of the blood anticoagulant was 0.5 EU in the reaction solution.
/ Ml or less.

The amount of the blood anticoagulant varies depending on the amount of blood collected, but usually 0.5 to 5 mg / ml for sodium ethylenediaminetetraacetate and 3 to 5 mg / ml for sodium citrate in the blood. % Of heparin sodium is used in an amount of 4 to 50 U / ml. Therefore, the endotoxin content of the blood anticoagulant of the present invention is appropriately selected depending on the amount of the blood anticoagulant and the collected blood volume, and the endotoxin content in the finally collected blood is 0.5.
It is preferably at most EU / ml. For example, when 1 ml of blood is collected and tested using sodium heparin, since heparin sodium is added at 4 to 50 U / ml, the endotoxin content is 0.125 EU / heparin unit or less, preferably 0.01 EU / heparin unit. /
Heparin unit or less.

The method for producing a blood anticoagulant having a low endotoxin content includes inactivation by heat treatment, acid or alkali treatment, ultrafiltration with a membrane filter,
Various known methods such as an anionic chitosan-based resin, polymyxin B that specifically binds to endotoxin, and a removal method using an adsorbent on which an antibody against endotoxin is immobilized can be used. However, heat treatment,
Inactivation by an acid or alkali treatment may be inactivated by some blood anticoagulants, so ultrafiltration with a membrane filter and removal with an adsorbent are preferred.

[Invention 5.6: Kit for Cell Function Measurement]
The kit for measuring cell function according to the fifth aspect of the present invention is configured by combining the cell function measuring container according to any one of the first to fourth aspects of the present invention with a reagent capable of quantifying the amount of a physiologically active substance. As an example of the method of using the cell function measurement kit, as described above, the method can be performed in the same manner as the method described in one embodiment of measuring the cell function using the cell function measurement container of the present invention 1.

The kit for measuring cell function of the present invention 6 comprises a container for measuring cell function of the above-mentioned present invention 2 (hereinafter referred to as a first measuring container) and a liquid in which the amount of endotoxin in the container is to be measured. The concentration in the extract when water equal to the amount was collected in the container and extracted was 0.5 EU /
It is constituted by combining a control value measurement container (hereinafter, referred to as a second measurement container) having a volume of not more than ml and a quantification reagent for quantifying a physiologically active substance.
In the kit for measuring cell function according to the present invention 6, the first
In the measurement container, the physiologically active substance in the blood is quantified, and in the second measurement container, the amount of the physiologically active substance in the blood not produced by the reaction with endotoxin (control value) is quantified. From the difference, an accurate amount of bioactive substance produced by endotoxin can be measured.

As the second measuring container, the present invention 2
The container before storing a sufficient amount of endotoxin for producing a physiologically active substance, which has been described in the structure of the cell function measuring container described above, can be used. That is, the amount of endotoxin in the container does not affect the measured value of the physiologically active substance, so that water equal to the amount of the liquid to be measured is collected in the container and extracted when the extraction is performed. As concentration
A container having a concentration of 0.5 EU / ml or less is used.
The method for extracting and measuring the endotoxin is the same as that of the present invention 1
The same method as that described for the cell function measurement containers of Nos. To 4 can be used.

The details of the first measuring container are as described in the cell function measuring container of the second embodiment of the present invention.

In order to measure the cell function using the cell function measuring kit according to the sixth aspect of the present invention, the blood collection container is connected to the first measurement container, and a blood sample is introduced into the first measurement container.
Next, the blood cells are reacted with endotoxin while appropriately shaking the first measurement container.

In order to obtain a control value, the blood collection container is communicated with the second measurement container, and the blood sample is guided to the second measurement container. In addition, blood anticoagulant is added in advance,
Blood can be directly collected in the second measurement container by using the decompressed second measurement container. In this case, the second measurement container and the first measurement container are connected, and the sample blood can be dispensed from the second measurement container to the first measurement container.

Next, as described above, the production of a physiologically active substance is induced in the first and second measurement vessels into which blood has been introduced, and thereafter, the blood cells and plasma are separated by standing or centrifuging. Then, the amount of the physiologically active substance in the plasma is determined using an enzyme immunoreagent or the like. At this time, the amounts of the physiologically active substances in the plasma in the first and second measurement containers may be separately measured using first and second enzyme immunoassay reagents having different measurement sensitivities.

When the reaction temperature between the blood and endotoxin in the first measurement container decreases, the metabolic activity of the cells decreases, and the amount of cytokine induction decreases. When the reaction temperature increases, the cells are damaged, and the amount of cytokine induction decreases. Therefore, it is preferably from 26 to 45 ° C, more preferably from 30 to 45 ° C.
42 ° C.

The reaction time between the blood and endotoxin is preferably 1 to 6 hours, more preferably 2 to 4 hours, which is a reaction time at which cytokine production is carried out efficiently and does not cause excessive hemolysis.

In the kit for measuring cell function according to the sixth aspect of the present invention, the amount of the physiologically active substance in the blood in the first measurement container, ie, the amount of the physiologically active substance in the blood produced by the reaction with endotoxin is determined by the first enzyme immunoreagent. The amount of the physiologically active substance in the blood that is not produced by the reaction with endotoxin is determined using the second enzyme immunoassay reagent. From these differences, an accurate amount of bioactive substance produced by endotoxin can be measured.

Usually, the amount of cytokines in the blood not reacted with endotoxin (ie, the amount of cytokines in the blood of the first measurement reactor) is from several pg / ml to several hundreds of p.
g / ml, and the amount of cytokine in the blood after reacting with endotoxin ranges from several hundred pg / ml to several thousand pg / m
1 or more.

However, several pg / ml to several thousand pg / ml
There is no cytokine measurement reagent having a wide measurement sensitivity of ml. Therefore, when measuring the amount of cytokines of 1,000 pg / ml or more, the plasma had to be diluted with an appropriate diluting solution and measured. However, the operation of diluting the plasma is complicated and requires a diluent and a dilution container separately, which significantly increases the time required for measurement. In addition, since the measured value is obtained by multiplying the dilution factor, there is a problem that the accuracy of the measured value is reduced.

On the other hand, in the present invention 6, the amount of cytokines in the blood not reacted with endotoxin (ie, the amount of cytokines in the blood in the second measurement container) can be measured, for example, at a measurement sensitivity of 10 to 1,000 pg / ml. And the amount of cytokine in blood reacted with endotoxin (ie, the first enzyme immunoassay)
The amount of cytokine in the blood in the measurement container) can be measured with a second enzyme immunoassay reagent having a low sensitivity such as a measurement sensitivity of 500 to about 10,000 pg / ml.

Therefore, the complicated dilution operation as described above is not required. In addition, the cytokine is TNF-
In the case of α and IL-1β, the sensitivity of the first enzyme immunoassay reagent is preferably 10 to 500 pg / ml, and the sensitivity of the second enzyme immunoassay reagent is preferably 500 to 10,000 pg / ml. In the case of IL-6, the sensitivity of the first enzyme immunoassay reagent is 10 to 1,000 pg / ml, and the sensitivity of the second enzyme immunoassay reagent is 1,000 to 20,000 p.
g / ml is preferred.

The first and second enzyme immunoassay reagents are composed of monoclonal or polyclonal antibodies against the cytokine to be measured, enzymes such as peroxidase and alkaline phosphatase, and a chromogenic substrate of each enzyme. A sandwich enzyme immunoassay in which a monoclonal antibody against a cytokine to be measured is previously immobilized on a solid phase surface such as a microplate is preferable because it is not necessary to immobilize the antibody before measurement and the reproducibility is excellent.

The method for immobilizing the monoclonal antibody on the surface of the solid phase may be any known method such as a physical adsorption method or a chemical bonding method, but the physical adsorption method is preferred because the operation is simple. The first and second enzyme immunoassay reagents having different assay sensitivities can be achieved by appropriately adjusting the monoclonal antibody or polyclonal process for cytokines and the concentrations of enzymes such as pen oxidase and alkaline phosphatase and the color-forming substrate of each enzyme. I can do it.

For example, when the sandwich enzyme immunoassay method is used, the first and second enzyme immunoassay reagents having different sensitivities can be prepared by adjusting the amount of the monoclonal antibody to the cytokine immobilized on the microplate. . That is, the amount of the target cytokine that can bind to the monoclonal antibody changes according to the amount of the monoclonal antibody immobilized on the surface of the microplate, so that reagents having different measurement sensitivities can be prepared.

Further, a reagent for detecting a cytokine bound to the immobilized monoclonal antibody, that is, an enzyme-labeled monoclonal antibody or an enzyme-labeled polyclonal antibody for a cytokine to be measured different from the immobilized monoclonal antibody is used at a different concentration. It is also possible by preparing.

Also, a method of incorporating a specific binding mode such as avidin-biotin into the above-mentioned enzyme immunoassay system, for example, using a biotin-labeled antibody instead of the above-mentioned immobilized monoclonal antibody instead of an enzyme-labeled antibody against a cytokine to be measured. There is a method using an antibody and further using an avidin-labeled enzyme.

[Invention 7: Method for measuring cell function] Inventive 7
The method for measuring a cell function according to the above is characterized in that blood is introduced into the cell function measuring container according to the present invention, and the cell function is measured. In this case, preferably as described above,
A blood anticoagulant whose endotoxin content during blood anticoagulation is limited to an amount that does not produce a physiologically active substance from blood cells when mixed with blood is previously contained in a cell function measurement container. One embodiment of the cell function measuring method according to the seventh aspect of the present invention will be described below. 100 EU of endotoxin and 10 U of heparin sodium are added, and 1 mL of blood is collected from the subject into a cell function measurement container which is stoppered under reduced pressure so that 1 mL of blood can be collected. Subsequently, the cells are incubated at a temperature of 26 to 45 ° C. for 1 to 6 hours to induce the production of TNF-α from blood cells, particularly monocytes and macrophages. Next, this blood is centrifuged at 1600 G, and the amount of TNF-α produced in the plasma is measured by an enzyme immunoassay.

[0080]

Next, the present invention will be described in detail with reference to examples and comparative examples, but the present invention is not limited to these examples.

In the following Examples and Comparative Examples,
Glass instruments and containers are subjected to a dry heat treatment at 250 ° C. for 2 hours, and plastic instruments and containers are either commercially available endotoxin-free or previously immersed in a 0.2 M aqueous sodium hydroxide solution overnight to obtain endotoxin. Was inactivated and washed thoroughly with endotoxin-free water. The operation was performed in a clean bench.

(Examples 1 to 4, Comparative Examples 1 to 5) (1) Production of Cell Function Measurement Container A polyethylene terephthalate 4 ml blood collection tube (diameter 12.6 × 75 mm) was used in endotoxin-free water (Otsuka Pharmaceutical Co., Ltd.) Wash well 4 times with 4 ml, 200 U
/ Heparin sodium (manufactured by Novo Nordisk A / S, trade name Novo Heparin Note 1000)
Physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) containing
05 ml was added. Further, E. coli UKT-B-derived endotoxin (Japanese Pharmacopoeia standard Lot. 892)
0) was dissolved in physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and serially diluted, and two heparin-containing blood collection tubes were added to each of the two blood collection tubes. The endotoxin concentration in the solution was 0 EU / ml (Comparative Example 1), 0.2 EU / ml
(Comparative Example 2), 0.5 EU / ml (Comparative Example 3), 0.8
EU / ml (Example 1), 8 EU / ml (Example 2),
80 EU / ml (Example 3), 800 EU / ml (Example 4), 8000 EU / ml (Comparative Example 4) and 8000
0.05 ml was added so as to be 0 EU / ml (Comparative Example 5), and dried under reduced pressure at room temperature.

Then, a well-washed endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd.) was gently placed on the opening of the blood collection tube so that the opening was not sealed, The container was placed in a container capable of reducing pressure, and the container was further reduced in pressure. When the pressure was reduced to 570 mmHg, the opening of the blood collection tube was sealed. The vacuum blood collection tubes manufactured in this manner were used as containers for measuring cell functions of Examples 1 to 4 and Comparative Examples 1 to 5.

(2) TNF-α, IL-1β, IL-6
Method of measuring the inducibility of healthy volunteers from a healthy volunteer to a syringe with a needle
Heparin blood was collected by each ml, and the injection needle was pierced into the butyl rubber plug of the vacuum blood collection tube to which various concentrations of endotoxin obtained in the above (1) were added, and 0.9 ml of the sample blood was injected into the blood collection tube. Collected. Next, 37
Each reduced-pressure blood collection tube from which blood was collected was attached to a rocker platform for inversion mixing in an incubator kept at a temperature of ° C., and mixed by inversion for 4 hours. After mixing, each reactor was centrifuged at 1600 G for 10 minutes at 4 ° C., and the supernatant plasma was collected. TNF-α, IL-1 in collected plasma
β and the amount of each cytokine (pg / ml) were measured using an enzyme immunoassay kit using each monoclonal antibody. The measurement of the amount of TNF-α production (induction) was performed using Genzyme Corporation, trade name “Predicta Human TNF-α ELISA KIT (detection limit 35 pg / ml)”. In addition, IL-
The measurement of the amount of 1β production (induction) was performed using Genzyme's trade name “Predicta Human IL-1β ELISA KIT”. The detection limit concentration of this measurement method is 15 pg / ml.
Met. Five measurements were made for each.

(Results) The above measurement results are shown in FIG. 1 and Table 1. FIG. 1 shows the concentration of TNF-α and IL-1β in plasma with respect to LPS concentration (EU / ml) (endotoxin concentration in blood (in whole solution) upon contact with blood). The average of the values obtained from Table 1 is shown. Also,
Table 1 shows the CV values (= standard deviation / average value × 100) of the values obtained from each of the five blood collection tubes for the concentrations of TNF-α and IL-1β in plasma in the same manner as described above. From the above results, it was found that when the endotoxin concentration was 0.6 EU / ml or more, induction of TNF-α and IL-1β production occurred. In addition, the endotoxin concentration is 8 to 800
In EU / ml, the induction amounts of TNF-α and IL-1β showed almost equal values. And, up to 8000 EU / ml, TNF-α,
The amount of induction of IL-1β is increasing,
In the case of U / ml, the amount of induction is smaller than that in the case of 8000 EU / ml. In addition, the endotoxin concentration is 800
At EU / ml or less, the dispersion of the measured values was very small, and accurate measurement was possible. However, 8000 EU / ml
Above, the dispersion of the measured values became large.

[0086]

[Table 1]

(Example 5, Comparative Example 6) Novoheparin (1000 U / mL) was collected to a 50 mL syringe for blood collection (manufactured by Terumo Corporation) so that the concentration became 10 to 20 U / 1 mL of blood, and the injection needle was used. Was replaced with a 21G pterygoid needle, and 45 mL of blood was collected from the elbow vein of one healthy person (KK). Container for control value measurement (endotoxin amount 0.1 EU / tube), low-dose LPS reaction tube (100
EU / tube (Example 5)), high-dose LPS reaction tube (50,
000EU / mL tube (Comparative Example 6))
Each L was dispensed (using an LPS-free chip). The cells were immediately cultured at 37 ° C under shaking and mixing conditions (10 times / min). 1, 2, 4, 8, 24 hours after the start of the reaction, 4 ° C., 1600
After centrifugation at G, the supernatant plasma was dispensed into Eppendorf tubes and stored frozen at -80 ° C. The cryopreserved plasma was thawed at room temperature, and the TNF-α concentration in the plasma was measured using an EIA reagent for TNF-α measurement.

(Results) The above measurement results are shown in Table 2. Table 2 shows the relationship between the TNF-α production reaction time and the amount of TNF-α produced, and the TN obtained from each of the three reaction vessels is shown in Table 2.
It was represented by the average value of F-α production and its standard deviation.

[0089]

[Table 2]

As is clear from Table 2, the amount of TNF-α produced by the low-dose LPS stimulation (Example 5) reached a plateau in 4 hours. On the other hand, in the high-dose LPS stimulation (Comparative Example 6), the amount of TNF-α production increased up to 8 hours, and at least
It was believed that the plateau reached after 8 hours. From these results, the endotoxin concentration in the whole solution was 0.6 to 1000
It can be concluded that low-dose LPS stimulation at EU / ml can provide more accurate measurement in a shorter time.

(Example 6, Comparative Example 7) Examination of usefulness as an immunological function test method in an immunological abnormality model animal Five 7-week-old Lewis male rats were preliminarily reared for one week, and then 6 mg / ml Mycobacterium. tubercurosis H37RA
An adjuvant solution of liquid paraffin (pharmacopoeia) was administered subcutaneously to the right hind paw. Adjuvant solution is Mycobacter
ium tubercurosis H37RA powder was crushed with a pestle and agate mortar, crushed while adding liquid paraffin little by little to a uniform suspension of 20 ml, transferred to a glass test tube (50 ml) with a screw lid, and heated to 121 ° C. And 2
Autoclave sterilize for 0 minutes, sonicate well,
A homogeneous suspension was used. Plethysm
An adjuvant arthritis was monitored by measuring with an ometer (manufactured by Ugo Basile).

From the rats to which the adjuvant had been administered, the blood of each rat was injected from the jugular vein before administration of the adjuvant, and on days 8, 15 and 23 after administration with heparin Na (8 to 10un).
2 mL of blood was collected using a 2.5 mL syringe (manufactured by Terumo Corporation) and a blood collection needle using 22G. Immediately after collecting the collected blood (0.2 mL), the blood was placed in an Eppendorf tube to which EDTA-2K had been added in advance to a final concentration of 1 mg / ml, and diluted twice with physiological saline. (Sysmex) to measure the white blood cell count.
Further, 0.8 ml of the collected blood was diluted 2-fold with an RPMI1640 medium containing an antibiotic, and used for inducing TNF-α production.

TNF-α production was induced by LPS (E. coli.
5) 0.5 mL each of the diluted blood collected was added to the previously dried reaction tube after adding 100 EU (Example 6) and 50,000 EU (Comparative Example 7), and gently stirring at 37 ° C. for 4 hours. Culture was performed. Thereafter, the plasma was centrifuged, and the amount of TNF-α produced in the plasma was measured with an ELISA kit (Genzyme). In addition, the lymphocyte blastogenesis ability was measured by a microwashed whole blood method.

(Results) The above measurement results are shown in FIGS. FIG. 2 shows the correlation between the amount of TNF-α produced by low-dose LPS (200 EU / ml) stimulation and lymphocyte blastogenesis, and FIG. 3 shows the high-dose LPS (50,000 EU / m2).
1) Correlation between the amount of TNF-α produced by stimulation and the lymphocyte blastogenesis ability is shown. As a pathological model causing abnormalities in the immune system, a correlation was observed between the ability of TNF-α production and lymphocyte blastogenesis induced by low-concentration LPS stimulation in rats that developed adjuvant arthritis (p = 0.
On the other hand, no correlation was observed with the high concentration LPS stimulation. As a result, it can be said that the method using low-concentration LPS can more specifically detect abnormal immune function.

(Example 7) Investigation of specificity of monocytes and macrophages 50 m from the elbow vein of a healthy volunteer using a syringe to which heparin was previously added so as to be 10 to 20 U / ml.
1 was bled. LPS storage capacity is 675.3
39.123.53.24.5.5 and 0.1 (EU /
10 μg / Breferdine A to 7 test reaction tubes
0.02 ml was added, and heparinized blood collected from the above-mentioned healthy persons was dispensed in 1 ml portions, and reacted at 37 ° C. for 4 hours. Next, the FastImmune Cytokine System (BECTON DICKI
According to the protocol of NSON, 20 μl of PE-labeled mouse anti-human LeuM3 monoclonal antibody was added to each of the capped polystyrene centrifuge tubes. Next, 100 μl of the blood after the above operation was added thereto, followed by stirring for 3 seconds. The centrifuge tube was wrapped with aluminum foil and reacted at room temperature for 15 minutes. Thereafter, FACS Lysing So
2 ml of lution was added and vortexed for 3 seconds. The centrifuge tube was wrapped with aluminum foil and reacted at room temperature for 10 minutes. Then
After centrifugation at 500 G for 5 minutes, the supernatant was removed without aspirating the pellet. Then, FACS Permeabilizing Soluti
On was added in an amount of 500 μl, vortexed for 3 seconds, the centrifuge tube was wrapped with an aluminum foil, and reacted at room temperature for 10 minutes. Next, 4 ml of a washing solution (0.5% BSA-PBS) was added, and the mixture was centrifuged at 500 G for 5 minutes, and the supernatant was removed without sucking the pellet. Next, FITC-labeled mouse anti-human TNF-α monoclonal antibody and FITC-labeled mouse IgG1 at the same concentration were
Add, stir for 3 seconds, wrap the centrifuge tube with aluminum foil,
The reaction was performed at room temperature for 30 minutes. Next, 4 ml of the washing solution
The mixture was centrifuged at 500 G for 5 minutes, and the supernatant was removed without aspirating the pellet. Then, 500 μl of 1% paraformalmaldehyde was added and stirred for 3 seconds.
This sample was stored in a cool dark place, and the measurement was performed using a flow cytometer (EPICS-XL) within 24 hours. FS
Lymphocytes, monocytes, and granulocytes were fractionated by / SS. lymphocytes,
Intracellular TNF-α-positive regions of the monocyte and granulocyte fractions were identified as FI ≧ 1 from the results of FITC-labeled mouse IgG1 staining.
0, FI ≧ 10, FI ≧ 30, and the proportion of intracellular TNF-α positive cells in each fraction was evaluated.

(Results) The above measurement results are shown in Table 3. Intracellular TNF-α-positive cells are mostly found only in CD14-positive monocyte and macrophage fractions,
It can be concluded that -alpha producing cells are monocytes and macrophages. From these results, if the concentration of endotoxin in the whole solution is in the range of 0.6 to 1000 EU / ml, the TNF-α-producing ability can be used as an index even if monocytes and macrophages are not separately separated from whole blood. It has been shown that a highly specific monocyte function test can be performed.

[0097]

[Table 3]

[0098]

Since the present invention has the above-mentioned structure, it is possible to accurately measure the ability of blood cells, particularly leukocytes, to produce physiologically active substances such as cytokines without causing unnecessary activation of cells. it can. In addition, whole blood can be used as it is, and there is no need to separate monocytes and lymphocytes from blood, which simplifies the operation and eliminates the problem of reduced activity accompanying the separation operation. Function can be measured. Furthermore, after collecting blood from the subject, it is not necessary to transfer blood to various measuring containers by means such as pipetting, or to perform operations such as cell separation and cell culture. In addition, there is almost no risk of being infected with the infectious disease, the measurement time can be reduced, and expensive equipment such as an RI facility or a flow cytometer is not required. In addition, the function of monocytes and macrophages in blood cells can be specifically measured.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of measuring the amount of TNF-α and IL-1β induction at each LPS concentration using the cell function measurement containers obtained in Examples 1 to 4 and Comparative Examples 1 to 5.

FIG. 2 is a graph showing the correlation between the amount of TNF-α production and lymphocyte blastogenesis by low-dose LPS (200 EU / ml) stimulation.

FIG. 3 is a graph showing the correlation between the amount of TNF-α production and lymphocyte blastogenesis by high-dose LPS (50,000 EU / ml) stimulation.

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Sumi Kuriyama 2-1 Hyakuyama, Shimamoto-cho, Mishima-gun, Osaka Sekisui Chemical Co., Ltd.

Claims (7)

[Claims] 1. A container for measuring a cell function, which is used for measuring a physiologically active substance produced by blood cells, wherein the amount of endotoxin in the container in advance does not affect the measured value of the physiologically active substance. In the container specified in, a sufficient amount of endotoxin to cause blood cells to produce a physiologically active substance is stored in a state in which the endotoxin can come into contact with the collected blood. A cell function measurement container, wherein the concentration in the liquid is 0.6 to 1000 EU / ml. 2. A container for measuring a cell function, which is used when measuring a physiologically active substance produced by blood cells, wherein the amount of endotoxin in the container is equal to the amount of liquid to be measured in advance. The concentration in the whole liquid when it is brought into contact with blood is 0.6 to 1000 in a container having a concentration of 0.5 EU / ml or less as the concentration in the extract when it is collected and extracted.
A cell function measurement container, wherein endotoxin is housed in a state where the endotoxin can be brought into contact with the collected blood so as to have EU / ml. 3. The container for measuring cell function according to claim 1, wherein the container comprises a bottomed tube having one end opened and the other end closed, and a blood sample is provided at an opening of the container. A cell function measurement container, which is provided with a plug that is hermetically sealed without being removed at the time of collection. 4. The container for measuring cell function according to claim 3, wherein the inside of the container is evacuated. 5. A cell function measurement container, comprising: the cell function measurement container according to claim 1; and a reagent capable of quantifying the amount of the induced physiologically active substance. kit. 6. A container for measuring cell function according to claim 2, and an extract obtained by extracting water in which the amount of endotoxin in the container is equal to the amount of the liquid to be measured and extracting the water. A cell function measurement kit comprising: a control value measurement container having a medium concentration of 0.5 EU / ml or less; and a quantification reagent for quantifying a physiologically active substance. 7. A method for measuring cell functions of monocytes and macrophages, wherein the kit for measuring cell functions according to claim 5 or 6 is used.