JPH11118799A - Method, kit and reactor for measuring cell function - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method, a kit and a reactor for measuring cell function accurately in safety through a simpler operation. SOLUTION: A material (e.g. combination of endtoxin and lectin, e.g. phytohemaglutinine) for inducing production of lymphokines (e.g. interleukin-2) through contact with blood and a coagulant (e.g. heparin sodium) are placed in a pressure reduced container such that they can touch blood. Quantity of endtoxin in a nonused container is limited to have no effect on the measurement of lymphokines.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cell function measurement reactor, a cell function measurement method, and a cell function measurement kit used for measuring cell functions related to an immune reaction, particularly for measuring lymphocyte functions.

[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 reaction and an immune reaction in blood and in various organs and organs. These cells play important roles in various disease states such as infectious diseases; inflammatory diseases such as hepatitis and nephritis; immunological and allergic diseases such as rheumatoid arthritis and asthma; and cancers. Is known to suppress or enhance the function of cells.

[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 state 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, in order to measure such a cell function, a granulocyte phagocytosis function test, a granulocyte bactericidal ability (active oxygen producing ability) test, a lymphocyte blastogenesis test and the like have been performed. In recent years, surface antigen tests using a flow cytometry device and a fluorescently labeled monoclonal antibody against various immunocompetent cell surface antigens have been performed. However, conventional test methods include cell isolation, cell culture,
Special techniques such as microscopic measurement were required, the measurement took time, and expensive equipment such as RI facilities and flow cytometry was required.

Various methods have been reported so far for examining the function of producing cytokines from blood or leukocytes separated from blood. For example, Japanese Patent Publication No. 1-503331,
JP-A-2-196951, JP-A-3-28569
No. 2 discloses that tumor necrosis factor α (TNF) produced by reacting blood with lipopolysaccharide (LPS) or lectin.
α), a method for measuring cytokines such as interleukin-1β (IL-1β) has been disclosed.

[0006] 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 or the method disclosed in the above-mentioned patent publication. That is, in these tests, after collecting blood from a subject with a syringe, blood is transferred to various reaction vessels by means of a pipetting method, or cell separation for separating white blood cells, Special operations such as cell culture for function 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. Also, during these operations, various germs and dust may be mixed into the blood sample,
These contaminants or physical stimuli due to manipulation may unnecessarily stimulate the cells in the blood, which may adversely affect the measurement results.

Further, even if blood cells are stimulated with LPS or lectin alone, interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-5
Lymphokines such as (IL-5) and interferon-γ (INF-γ) have the disadvantage that only small amounts are produced.

[0008] Some commercially available blood collection tubes also contain endotoxins such as LPS derived from Gram-negative bacteria, and the endotoxin content varies between lots. Therefore, even if a certain amount of LPS and lectin are added as a stimulant, the difference between the lots of the endotoxin content in the blood collection tube remains as it is, so that the amount of lymphokine produced differs depending on the lot. Can not solve.

[0009] Because of these problems, there is a demand for a method for measuring cell functions that is simpler, less dangerous, and more accurate than before.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to eliminate the drawbacks of the conventional cell function measuring method, and to make the operation easier and less dangerous than the conventional method for measuring cell functions. It is an object of the present invention to provide a device, a method for measuring cell function, and a kit for measuring cell function.

[0011]

A reactor for measuring cell function according to claim 1, wherein a material which induces the production of lymphokines in the blood by contacting the blood with a vessel having a reduced pressure inside. In addition, the amount of endotoxin in the container before use is restricted so that the blood anticoagulant can be brought into contact with blood and does not affect the measured value of lymphokine.

The reactor for measuring cell function according to claim 2 is
The amount of endotoxin in the container before use is 0.5 EU / ml or less as the concentration in the extract when the amount of endotoxin-free water equal to the amount of the liquid to be measured is collected and extracted. The cell function measuring reactor according to claim 1, wherein the materials that induce lymphokine production are endotoxin and lectin.

The reactor for measuring cell function according to claim 3 is
Lectin is phytohemagglutinin, concanavalin A
3. The reactor for measuring cell function according to claim 2, wherein the reactor is at least one member selected from the group consisting of: and porkweed mitogen.

[0014] The reactor for measuring cell function according to claim 4 comprises:
The concentration of endotoxin in the whole solution when brought into contact with blood is 0.6 to 100,000 EU / ml, and the concentration of phytohemagglutinin is 0.01 to 100 μg / ml. 4. The reactor for measuring cell function according to item 3.

The method for measuring cell function of the present invention comprises
The blood is sucked into the cell function measurement reactor according to any one of the above items 4, and is brought into contact with the material that induces the production of lymphokines to induce lymphokines.

[0016] The kit for measuring cell function of the present invention is described in claim 1.
The reactor for measuring cell function according to any one of to 4,
And a reagent capable of quantifying the amount of the induced lymphokine.

Hereinafter, the reactor for measuring cell function of the present invention will be described. The lymphokine-inducing material used in the present invention refers to a material that reacts with lymphocytes and the like in blood, promotes the activation of these cells, and induces the above-described lymphokine. Although various materials known as are used, it is preferable to use endotoxin and lectin in combination. By using endotoxin and lectin in combination as an inducing material, there is an advantage that the production of lymphokines from lymphocytes can be induced in a shorter period of time than when each is used alone.

The above-mentioned endotoxin mainly refers to LPS derived from Gram-negative bacteria. Instead of this endotoxin, it is composed of various microorganisms such as killed BCG and Corynebacterium, and cell wall polysaccharide (lipopolysaccharide) derived from microorganisms. It may be used in combination from the group of endotoxin, synthetic lipid A, pyran copolymer and the like. In addition, phytohemagglutinin (P
HA-L and PHA-P), at least one selected from the group consisting of concanavalin A and pork weed mitogen. Poly I: poly C; purified protein-free tuberculin; OK432 (Picibanil); PSK
(Crestin); Lentinan; Zymozan; Interferon; Stimulators such as interleukin-1 (IL-1), interleukin-6 (IL-6), TNF and other cytokines; . In addition, materials obtained by immobilizing these materials on various natural or synthetic polymer materials by a known immobilization method may be used.

In the reactor for measuring cell function of the present invention,
The amount of the endotoxin used is such that when it is brought into contact with blood, the concentration of endotoxin in the whole liquid (total of blood, blood anticoagulant solution, endotoxin solution and lectin solution) is 0.6 to 100000 EU / ml. It is preferably set to be 0.8 to 80000 EU
/ Ml is more preferable.
If the concentration is less than 0.6 EU / ml, the amount of lymphokine induction decreases, and if it exceeds 100,000 EU / ml, an increase in the amount of induction cannot be expected and the cost increases.

In the reactor for measuring cell function of the present invention,
The amount of lectin used in combination with the above endotoxin is such that the lectin concentration in the whole solution (the sum of blood, blood anticoagulant solution, endotoxin solution and lectin solution) when brought into contact with blood is 0. .01-100 μg /
ml, preferably 0.1 to
More preferably, it is adjusted to 80 μg / ml. If the concentration is less than 0.01 μg / ml, the amount of lymphokine induction decreases, and if it exceeds 100 μg / ml, an increase in the amount of induction cannot be expected and the cost increases.

In the cell function measuring reactor of the present invention, the above-mentioned lymphokine-inducing material is in a state in which the inside is evacuated to be in contact with blood. Examples of the state in which the blood can be contacted include a case where the lymphokine-inducing material is contained in the container.

The above-mentioned lymphokine-derived material is usually in the form of powder or liquid in which the derived material is dissolved in a solvent such as water. Further, any known shape such as a fibrous shape, a hollow fiber shape, and a film shape may be used. The state of the induction material in the container may be a solid state,
It may be a gel or a liquid. When the guide material is water-soluble, it is dissolved in an appropriate solvent and applied to the inner wall surface of the container.
Alternatively, it may be made into a powder after being added.

Examples of the solvent include buffers such as phosphate buffer and Hanks buffer, MEM, RPMI-
Normal medium such as 1640 or water for injection (LPS-free water,
And Otsuka Pharmaceutical Co., Ltd.) and physiological saline.

The lymphokines referred to in the present invention are physiologically active substances produced from lymphocytes when stimulated with the above-mentioned stimulants, and include, for example, IL-2, IL-4, IL-
5. INF-γ, Granulocyte Macrophage Colony Stim
ulating Factor (GM-CSF), etc.
In particular, it is not limited to these.

Since the cell function measuring reactor of the present invention is used for measuring cell functions in blood, it is necessary that a blood anticoagulant is contained in the above-mentioned container so that blood does not coagulate.

The blood anticoagulant may be present in the container in either a liquid or solid state.

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.

The amount of heparin sodium stored in the container may be such that when blood is stored in the container, if the concentration of heparin sodium in the whole liquid containing the blood decreases, blood coagulation may occur. In such a case, unexpected activation or inactivation may occur in the cells.
It is preferable to accommodate them so as to be 1.

The material of the container of the reactor of the present invention is, for example, plastic or glass, and has a strength enough to withstand a centrifugal separation operation for measuring the lymphokine after the induction reaction of the lymphokine. Is preferred. As the plastic, a thermoplastic resin,
Any of the thermosetting resins can be used. As the thermoplastic resin, for example, polyethylene, polypropylene, polystyrene, polymethyl methacrylate, polyvinyl chloride, polyethylene terephthalate, styrene-acrylonitrile copolymer, styrene-maleic anhydride copolymer, styrene-acrylic acid copolymer, styrene -Methyl methacrylate copolymer, ethylene-propylene copolymer, ethylene-acrylic acid copolymer, ethylene-acrylic ester copolymer and the like. Examples of the thermosetting resin include an unsaturated polyester resin, an epoxy resin, and an epoxy-acrylate resin.

In the reactor of the present invention, a plug is usually used to maintain the inside of the reactor at a reduced pressure. Examples of the material of the plug include butyl rubber, chlorinated butyl rubber, and thermoplastic elastomer.

The degree of pressure reduction in the reactor of the present invention is determined by comparing the blood whose cell function is to be measured at normal pressure (hereinafter, the blood whose cell function is to be measured is sometimes referred to as a sample blood) with the blood described above. When the reactor is connected to the reactor, the pressure may be such that normal-pressure sample blood can be sucked into the reactor, 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.

The amount of blood used for measuring the cell function using the reactor of the present invention varies depending on the volume of the reactor. Usually, when a reactor having a volume of 4 to 5 ml is used,
It may be about 0.5 to 2 ml.

The endotoxin content of the container used in the present invention must be restricted before use so as not to affect the measured value of the production-induced lymphokine. If the container before use is contaminated with endotoxin, it may cause measurement variations. As is clear from the comparative examples described later, when the amount of endotoxin such as LPS increases, the above-mentioned lymphokine induction is remarkably caused. Therefore, in order to accurately measure the cell function according to the present invention, the endotoxin content must be below the level that does not cause the above-described lymphokine induction. The mechanism of the induction of lymphokines by endotoxin is that endotoxin significantly induces the induction of cytokines such as TNFα, IL-1β, and interleukin-6, and the stimulation of the cytokines causes IL-2, IL-4, I-4
Lymphokines such as NF-γ are unnecessarily induced. Therefore, in order to accurately measure the cell function according to the present invention, the endotoxin content must be adjusted to the above-mentioned TNFα, IL
It must be restricted to a level that does not cause induction of cytokines such as -1β and interleukin-6.

The above endotoxin content is determined by extracting an amount of endotoxin-free water equal to the amount of the liquid to be measured into the container and extracting the solution under stirring at 37 ° C. for 30 minutes. Is preferably 0.5 EU (international endotoxin unit) / ml or less [The amount of endotoxin-free water at the time of performing the above extraction is completely different from the amount of liquid to be measured as described above. It is not necessary that the amount be equal, and if the extraction is sufficiently performed, the amount may be less than the amount to be measured. However, even in this case, the endotoxin content in the extract is 0.5 EU / ml or less. Then, the effect of the present invention is exhibited.

The endotoxin is usually 220 to 2
It can be inactivated by dry heat sterilization at 50 ° C,
If dry heat sterilization is not possible, it can be reduced by washing with endotoxin-free water.

There are various methods for measuring the endotoxin content. The method for measuring the endotoxin content in the present invention is based on the synthetic chromogenic substrate method.

As an example of the method for producing the reactor of the present invention, the above-mentioned lymphokine-inducing material and blood anticoagulant were added to a container capable of reducing the pressure inside, and the container was brought into a predetermined reduced pressure state. Thereafter, the container is manufactured by plugging the container.

As the above-mentioned container, for example, a bottomed tubular body having one end opened and the other end closed is preferable.
What can be closed by a stopper is preferred. As the bottomed tube, for example, a test tube suitable for centrifugation operation for measuring the amount of lymphokine after the induction reaction of lymphokine is more preferable, and the outer diameter is 5 to 30 mm. , And a height of about 20 to 150 mm is preferable.

Further, the reactor of the present invention is preferably produced in a clean environment as much as possible in order to avoid contamination with endotoxins and various germs, and if possible, it is preferable to carry out a known sterilization treatment after the production. .

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

As the sample blood, besides the collected whole blood, if necessary, the collected whole blood may be, for example, ME.
M or a normal medium such as RPMI-1640 or a solution diluted with a solution that does not lyse blood, such as physiological saline, may be used.

As a method of connecting the blood collection container to the reactor, for example, a method of setting the blood collection container as a syringe with an injection needle and piercing the injection needle into the stopper of the reactor is mentioned.

When the above-mentioned blood vessel and the above-mentioned reactor are communicated with each other, a multiple injection needle used in a normal vacuum blood collection method, that is, a needle on one side of the needle base, which is a vascular piercing side, is provided. On the other side, a needle having a plug piercing side capable of piercing the plug of the reactor may be used, and a needle in which the blood vessel piercing side needle and the plug piercing side needle are communicated may be used.

The reaction temperature between the blood and the lymphokine-inducing material is a temperature at which the production of the lymphokine is carried out efficiently and does not cause excessive hemolysis.
C is preferable, and more preferably 30 to 40C. The reaction time is a reaction time at which the production of the lymphokine is performed efficiently and does not cause excessive hemolysis.
48 hours is preferred, more preferably 2 to 24 hours.

The method for measuring lymphokines induced by using the reactor of the present invention includes, for example, monoclonal antibodies or polyclonal antibodies against the above-mentioned lymphokines to be measured, enzymes such as peroxidase, alkaline phosphatase and the like. An enzyme immunoassay using a chromogenic substrate may be mentioned.

Hereinafter, one embodiment of measuring the cell function using the reactor of the present invention will be described in detail. First, the blood and the lymphokine-derived material are reacted in the reactor, and among INF-γ, IL-2 and IL-4,
At least one lymphokine is induced, and the reactor after induction is centrifuged at 1200 G to separate the blood cell component and the plasma component. Next, the separated plasma was added to the wells of the microplate on which the monoclonal antibody against lymphokine was immobilized by pipetting, and
Incubate at 7 ° C for 2 hours. Next, the plasma solution after the reaction is discarded by means such as suction removal, and further contains a nonionic surfactant such as Tween 20 to remove unreacted components.
Wash the wells with neutral pH wash buffer. Next, a polyclonal antibody against the above-mentioned lymphokine on which horseradish peroxidase is immobilized is added to the above-mentioned wells 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. Then
The reaction is stopped by adding a 1 M sulfuric acid solution, and the color of the substrate due to the enzymatic reaction is measured from the absorbance at 450 nm.
From the measured value and a calibration curve prepared using the known concentration of the lymphokine, the amount of the induced production of the lymphokine is measured.

Next, the cell function measuring kit of the present invention will be described. As the reagent used in the cell function measurement kit of the present invention, a reagent capable of quantifying the amount of the lymphokine induced using the cell function measurement reactor according to any one of claims 1 to 4. If not limited, examples thereof include, for example, monoclonal or polyclonal antibodies against lymphokines to be quantified, enzymes such as peroxidase and alkaline phosphatase, and reagents for enzyme immunoassay utilizing a chromogenic substrate of each enzyme. .

One of the methods of using the above-mentioned kit for measuring cell function is as follows.
As an example, the method is the same as the method described in the above-described embodiment in which the cell function is measured using the cell function measurement reactor of the present invention.

[0049]

Now, the present invention will be described in further detail with reference to Examples and Comparative Examples of the present invention. The present invention is not limited to the following examples.

Example 1 (1) Method for Manufacturing Cell Function Measurement Reactor Nine LPS-free blood collection tubes (12.6φ × 75 mm, manufactured by Sekisui Chemical Co., Ltd.) made of polyethylene terephthalate were prepared, and each of these blood collection tubes was prepared. , Heparin sodium (manufactured by Novo Nordisk A / S, trade name: Novo Heparin Note 1000)
0.05 ml of a 200 u / ml saline solution for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) in phytohemagglutinin-P (PHA)
-P) 0.05 ml of a 100 μg / ml saline (manufactured by Otsuka Pharmaceutical) solution (manufactured by DIFCO) and LPS
0.05 ml of a 20,000 EU / ml saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) solution (manufactured by LBL) was added.

Then, a butyl rubber stopper, which is well washed with endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd.) and fits the diameter of the tube, is gently placed on the opening of the blood collection tube so as not to tightly close the opening. The container was placed in a container that can be depressurized, and the container was evacuated to a reduced pressure. When the container was depressurized to 570 mmHg, the opening of the blood collection tube was sealed to produce the cell function measurement reactor of the present invention.

(2) Method for Measuring IL-2 Inducibility Heparin blood was collected from a healthy volunteer using a syringe equipped with a needle, and the needle was used for RPMI1640 medium (GI
Five LPS-free blood collection tubes each containing 3.2 ml (BCO) were stabbed, 0.8 ml of blood was added, and the blood was diluted 5-fold.

Next, the blood collection tube containing the blood diluted five-fold with the RPMI medium was placed in the above-mentioned (1) using a blood collection holder equipped with a multiple injection needle used for vacuum blood collection.
Was connected to the cell function measurement reactor obtained in the above, and 0.85 ml of 5-fold diluted blood was collected in the reactor. Next, the reactor was attached to a rocker platform for inversion mixing in a thermostat that had been kept at 37 ° C. in advance, and the mixture was inverted for 24 hours. After mixing, set the reactor at 3000 rpm,
After centrifugation at 4 ° C. for 10 minutes, the supernatant plasma was collected. The amount of induced IL-2 in the collected plasma was measured using an enzyme immunoassay kit (manufactured by Cayman, product number 583321) using a monoclonal antibody. Incidentally, the detection limit concentration of this measuring method was 1.5 pg / ml.

(3) Results of Measurement of IL-2 Inducibility Three healthy volunteers (volunteers A, B,
C) using the three cell function measurement reactors obtained in the above (1) per person, and performing the IL-2 inducibility measurement method in the above (2). Induction was performed, and the average, standard deviation, and coefficient of variation ((standard deviation / average) × 100) of the IL-2 concentration in the obtained plasma for each volunteer are shown in Table 1.

(4) Measurement of LPS content of blood collection tube L in blood collection tube of the same lot as blood collection tube used in (1) above
PS content was measured as follows. Endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd.) is collected in a syringe with an injection needle, and the injection needle is pierced into the butyl rubber stoppers of the three blood collection tubes of the same lot, and endotoxin-free water ( 1 ml of Otsuka Pharmaceutical) and 30
The mixture was stirred at 37 ° C for a minute to extract endotoxin. Next, the endotoxin content in this extract was measured by the synthetic chromogenic substrate method using Endospecy ES6 (trade name), an endotoxin measurement kit manufactured by Seikagaku Corporation. As a result, the endotoxin content in the extract was 0.5 EU / ml or less for all three tested blood collection tubes.

Comparative Example 1 Instead of the LPS-free blood collection tube used in Example 1, LP
Example 1 (1) to (3) except that a blood collection tube without S-free standard (SP-0705P, manufactured by Sekisui Chemical Co., Ltd.) was used.
Table 1 shows the average, standard deviation, and coefficient of variation ((standard deviation / average) × 100) of the obtained plasma IL-2 concentration for each volunteer.

(4) Measurement of LPS content in blood collection tube The LPS content in the blood collection tube of the same lot as the blood collection tube without the LPS-free standard used as described above was measured in the same manner as in Example 1 (4). The endotoxin content in the extract is 1
It was 10 EU / ml (average of the three blood collection tubes tested).

[0058]

[Table 1]

Examples 2 to 7 and Comparative Example 2 (1) Manufacturing method of cell function measurement reactor 27 LPS-free blood collection tubes (12.6φ × 75 mm, manufactured by Sekisui Chemical Co., Ltd.) made of polyethylene terephthalate were prepared.
Heparin sodium (manufactured by Novo Nordisk A / S, trade name: Novo Heparin Note 1000)
0.05 ml of a 200 u / ml saline solution for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added.

Using the blood collection tube containing heparin, the reactors for measuring cell functions of Examples 2 to 7 and Comparative Example 2 were manufactured as follows.

Container for Example 2 In each of the three blood collection tubes containing heparin, 0.05 ml of a physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) and LPS (L) were added.
0.05 ml of a 20,000 EU / ml saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) solution of BL Corporation) was added. -Container for Example 3 In each of the three blood collection tubes containing heparin, 0.05 ml of a physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) and LPS (L) were added.
0.05 ml of a 2000 EU / ml saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) solution of BL Corporation) was added.

Container for Example 4 In each of the six blood collection tubes containing heparin, 0.05 ml of a physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) and phytohemagglutinin-P (PHA-P) (manufactured by DIFCO) Of 1
A solution of 00 μg / ml physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added to 0.
05 ml was added. -Container for Example 5 0.05 ml of a physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) and phytohemagglutinin-P (PHA-P) (manufactured by DIFCO) were added to each of the three blood collection tubes containing heparin.
0 μg / ml saline (manufactured by Otsuka Pharmaceutical Co.)
5 ml was added.

Container for Example 6 Each of the three blood collection tubes containing heparin was provided with LPS (L
0.05 ml of a 20,000 EU / ml saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) solution of BL) and 100 μg of phytohemagglutinin-P (PHA-P) (manufactured by DIFCO)
/ Ml physiological saline (Otsuka Pharmaceutical) solution 0.05ml
Was added. -Container for Example 7 Each of the three blood collection tubes containing heparin was provided with LPS (L
0.05 ml of a 20,000 EU / ml saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) in 20,000 EU / ml, and 20 μg / ml of phytohemagglutinin-P (PHA-P) (manufactured by DIFCO).
0.05 ml of a physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added.

Container for Comparative Example 2 0.1 ml of a physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added to each of the six blood collection tubes containing heparin.

The containers for Examples 2 to 7 and the container for Comparative Example 2 prepared as described above were then washed well with endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd.), and a butyl rubber stopper fitted to the pipe diameter was used. Is placed lightly on the opening of the reactor so that the opening is not sealed, and then placed in a container that can be depressurized.
When the pressure was reduced to Hg, the opening of the container was sealed and
A reactor for measuring cell functions for Examples 2 to 7 and a reactor for measuring cell functions for Comparative Example 2 were produced.

(2) Method for Measuring IL-2 Inducibility and Results of Measurement Heparin blood was collected from a healthy volunteer using a syringe equipped with an injection needle, and the injection needle was placed in an RPMI1640 medium (GI
Seven LPS-free blood collection tubes each containing 3.2 ml (BCO) were pierced, and 0.8 ml of blood was added to dilute the blood 5 times.

Next, the blood collection tube containing the blood diluted 5-fold with the RPMI medium was placed in the above-mentioned (1) using a blood collection holder equipped with a multiple injection needle used for vacuum blood collection.
Of each of the reactors for measuring cell function for Examples 2 to 7 obtained in
This was connected to three of the cell function measurement reactors for Comparative Example 2 and Comparative Example 2, respectively.
5 ml were collected. Next, the reactor was attached to a rocker platform for inversion mixing in a thermostat that had been kept at 37 ° C. in advance, and the mixture was inverted and mixed for 24 hours.
Two inductions were performed. After mixing, set each reactor to 3000r
The supernatant was collected by centrifugation at 4 ° C. for 10 minutes at 4 ° C. The amount of IL-2 induction in the collected plasma was measured using an enzyme immunoassay kit (manufactured by Cayman, product number 583321) using a monoclonal antibody. Incidentally, the detection limit concentration of this measuring method was 1.5 pg / ml. Table 2 shows the average value, standard deviation, and coefficient of variation ((standard deviation / average value) × 100) of the obtained plasma IL-2 concentration in each of Examples and Comparative Examples.

(3) Measurement of LPS Content of Cell Function Measurement Reactor of Example 4 and Comparative Example 2 Of the cell function measurement reactors of Example 4 and Comparative Example 2 prepared in (1) above, LP in remaining three reactors not used for measurement of IL-2 inducibility in (2)
The S content was measured as follows. Endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd.) is collected in a syringe equipped with a syringe needle, and the injection needle is pierced into the butyl rubber stopper of each of the above-mentioned reactors. 0.85 ml was collected for 30 minutes, 3
The mixture was stirred at 7 ° C. to extract endotoxin. Next, the endotoxin content in this extract was measured using Endospecy ES6, an endotoxin measurement kit manufactured by Seikagaku Corporation.
(Trade name) using the synthetic chromogenic substrate method. As a result, the endotoxin content in the extract was 0.5 EU / ml or less for all of the six tested.

[0069]

[Table 2]

In Table 2, “all liquids in the concentration of lymphokine-derived material in all liquids at the time of induction reaction” refers to the amount of sodium heparin solution, the amount of LPS solution, the amount of PHA-P in the reactor at the time of induction reaction. It refers to the sum of the solution volume, physiological saline solution volume, and 5-fold diluted blood volume.

Tables 1 and 2 show the following. From Table 1, in the blood collection tube without the LPS-free standard (Comparative Example 1), the amount of IL-2 induction was higher than that in the case where the LPS-free blood collection tube was used (Example 1), and the variation (coefficient of variation). Was also very expensive. From this, the commercial LP
Blood collection tubes without the S-free standard are contaminated with LPS, and the LPS content is not constant. It is thought that the variation in the amount of lymphokine induction is large.

From Table 2, it can be seen that LP-2 was used as the IL-2 inducing material.
When S alone, PHA-P alone, LPS and PHA-P were used in combination, compared to the case where LPS and PHA-P were used alone, the case of using LPS and PHA together showed IL-2. The amount of induction was high. That is, in order to induce and measure lymphokines with high sensitivity, it is clear that it is better to use LPS and lectin together as stimulants. By using a cell function measurement reactor to which LPS and lectin are added as stimulants,
TNF produced mainly by stimulating monocytes and macrophages
The production amounts of α, IL-1β, and IL-6 can be measured, and lectins stimulate lymphocytes such as B and T cells to produce IL-2, IL-4, and INF.
There is also an advantage that the production of lymphokines such as -γ can be measured.

[0073]

The structure of the cell function measuring reactor according to the first aspect is as described above. When the cell function measuring reactor is used, blood is collected from a subject, and then pipetting is performed. By transferring blood to various reaction vessels by means of
Since no operation such as cell separation and cell culture is required, the tester has little risk of being infected with various infectious diseases such as hepatitis and AIDS. In addition, since the endotoxin content is controlled and there is no risk of contamination of various germs and dust in the blood sample, there is a problem that these contaminants and unnecessary activation or reduction of the activity of cells due to various operations are caused. Absent. In addition, since whole blood is used, no special techniques such as cell separation, cell culture, and microscopic measurement are required, and the measurement time can be reduced.
There is no need for expensive equipment such as I facilities and flow cytometry. Therefore, the operation is simpler and less dangerous than before, and the cell function can be measured with high accuracy.

The constitution of the reactor for measuring cell function according to claim 2 is that, as described above, the amount of endotoxin in the container before use is equal to the amount of the liquid to be measured. When the extraction is performed, the concentration in the extract is 0.5 EU / ml or less, and the materials for inducing the production of lymphokines are endotoxin and lectin. And the same effect as the cell function measurement reactor according to claim 1 can be obtained, and endotoxin alone,
Since the amount of lymphokine induction is higher than when lectin is used alone, the cell function can be measured with higher sensitivity.

According to the third aspect of the present invention, as described above, the lectin is at least one selected from the group consisting of phytohemagglutinin, concanavalin A, and pork weed mitogen. When the reactor for measuring cell function is used, all the effects of the invention described in claim 2 can be achieved, and the amount of lymphokine induction can be further increased, so that the cell function can be measured with higher sensitivity.

According to a fourth aspect of the present invention, as described above, the concentration of endotoxin in the whole solution when it is brought into contact with blood is 0.6 to 100000 EU / m.
l, and the concentration of phytohemagglutinin is 0.01 to 10
Since the concentration is set to 0 μg / ml, the use of the above-described cell function measuring reactor can achieve all the effects of the invention of claim 3 and particularly increases the amount of lymphokine induction, Cell function can be measured with high sensitivity.

As described above, the configuration of the cell function measuring method of the present invention is such that blood is sucked into the cell function measuring reactor according to any one of claims 1 to 4 to induce the production of the lymphokine. Since this is a cell function measuring method characterized by inducing lymphokines by contacting with a material to be treated, all effects of the inventions of claims 1 to 4 can be achieved by using this method.

The kit for measuring cell function according to the present invention comprises:
As described above, since the cell function measurement reactor according to any one of claims 1 to 4 and the reagent capable of quantifying the amount of the induced lymphokine are used, the operation is easier and more dangerous than before. And cell functions can be measured more easily and accurately.

Claims (6)

[Claims] 1. A container in which the inside is evacuated, a material that induces the production of lymphokines in the blood by contacting the blood, and a blood anticoagulant are brought into a state in which the material can contact the blood, and A cell function measurement reactor, wherein the amount of endotoxin in the container before use is limited so as not to affect the measurement value of the lymphokine. 2. The concentration of endotoxin-free water in the container before use is equal to the concentration in the extract when the amount of endotoxin-free water equal to the amount of the liquid to be measured is collected and extracted. 2. The reactor for measuring cell function according to claim 1, wherein the material for inducing production of lymphokines is 5 EU / ml or less, and endotoxin and lectin. 3. The reactor according to claim 2, wherein the lectin is at least one selected from the group consisting of phytohemagglutinin, concanavalin A and pork weed mitogen. 4. The concentration of endotoxin in the whole solution when contacted with blood is 0.6 to 100,000 EU / ml, and the concentration of phytohemagglutinin is 0.01 to 100 μg.
4. The reactor for measuring cell function according to claim 3, wherein the reactor is set to / ml. 5. A method for inducing lymphokine by inhaling blood into the cell function measuring reactor according to claim 1 and bringing the cell into contact with the material that induces the production of lymphokines. Cell function measurement method. 6. A cell function measurement kit comprising the cell function measurement reactor according to claim 1 and a reagent capable of quantifying the amount of induced lymphokine. .