JP2001083144A - Process for treating blood and composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply decide a cell reaction with good reproducibility by adding a chelating agent, an antiblood coagulation agent having no chelatability and a metal salt capable of eluting a bivalent metallic ion to a blood. SOLUTION: For example, an EDTA of a chelating agent and a calcium chloride as a metal salt are added to a blood to which a heparin is added as an blood anticoagulant, an allergen to be doubt in an examiner is added to the liquid, and a mediator such as a histamine or the like is released from a blood cell. The released histamine or the like is directly measured by a known measuring method. Here, as the allergen, for example, a pollen of a cedar or the like, a food such as an egg white or the like is used. As the used blood, a whole blood is preferred. As the chelating agent, a circuit acid, an oxalic acid or the like is additionally used. As the blood anticoagulant, a plasmin, a hirudine or the like is used. As the metal salt, an MgCl2, CaSO4 or the like is used. Thus, a cell function can be measured without separating the blood.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for treating blood cells by treating blood with a chelating agent, an anticoagulant having no chelating ability, and a metal salt capable of eluting divalent cations in an aqueous medium. The present invention provides a blood processing method and a blood processing composition that can determine a blood cell reaction with good reproducibility without separating the blood.

[0002]

2. Description of the Related Art Blood cells contained in blood are known to be erythrocytes, basophils, neutrophils, eosinophils, lymphocytes, monocytes, etc. These cells are necessary for living organisms. It is well known that they are involved in the transport of various oxygen and defense reactions of the living body such as the immune response and the inflammatory response.

[0003] The immune response and the inflammatory response are caused by the cell's own mediated phagocytosis, such as the phagocytosis of the blood cell itself, by the direct cell reaction to an enemy or tissue or the reaction of releasing the mediator held by the blood cell. It is known that it is performed by acting on other cells or tissues. As mediators released from cells (blood cells) in these blood, interleukins such as interleukin 1 (IL-1), interleukin 2
(IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (I
L-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-
8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 13 (IL
-13), interferon-α (IFN-α), interferon-β (IFN-β), interferon-
γ (IFN-γ), tumor necrosis factor (TNF-α, TNF
-Β) and other cytokines, histamine, leukotriene, platelet activatin
g factor (PAF), major basic
protein (MBP), eosinophil
Caticic protein (ECP), eos
inophil peroxidase (EPO), c
harcot-leyden crystal (CL
C) and the like are known. It is known that the release of these mediators from blood cells is sometimes excessive and causes symptoms such as bronchial asthma, rheumatoid arthritis, allergic rhinitis, atopic dermatitis and diabetes. Therefore, determining the amounts of these substances is very important in the field of examining the pathology of patients with the above-mentioned diseases, selecting drugs to be used for the treatment, and further, basic research on the diseases.

[0004] These conventional problems for measuring the amount of mediator in the blood include lack of reproducibility and an inconvenient operation. Mediators in blood are extremely small in amount, and it is known that abundant and complex proteins or blood cell components present in blood adversely affect the accuracy of measurement. In several steps, an operation of separating and measuring a mediator, other proteins, and blood cell components was required. Among the mediators, histamine and leukotriene are used as examples to explain the method of releasing a mediator from blood cells. The method for separating and measuring leukocytes from blood is described in Patent Publication No. 6-2.
05695. EDTA from subject to test tube
The collected blood and an antibasophil monoclonal antibody immobilized on magnetic particles are added, and the reaction is carried out at room temperature for 5 minutes to separate basophils from the blood. The magnetic particles are transferred to an avidin-immobilized microplate, and allergic specific antigen is dispensed into the wells of the microplate to release histamine from basophils. As a method for quantifying histamine, biotin-labeled histamine and enzyme-labeled antihistamine antibody were added to the reaction solution, and after washing, the enzyme activity of the biotin-labeled histamine-enzyme-labeled antihistamine antibody complex bound to the avidin-immobilized antibody was determined. A method for measuring the amount of histamine is disclosed.

As another method, in order to separate and measure leukocytes from blood, blood is collected under EDTA-added conditions, red blood cells are precipitated by adding 4% dextran, leukocyte suspension is separated, and centrifugation is performed. Perform tyroid reaction (1
24 mM NaCl, 4 mM KCl, 0.64 mM N
aH ₂ PO ₄ , 1 mM CaCl ₂ , 0.6 mM MgCl
₂ , 10 mM HEPES and 0.03% human serum albumin), centrifugation and suspension are repeated, and the leukocytes are washed. An allergic specific antigen solution is added to the suspension to cause a reaction, thereby releasing histamine from basophils. It is well known that after the reaction, centrifugation is performed, the supernatant is collected, and the amount of histamine released in the supernatant is measured by a fluorescence method, an RIA method, or an HPLC method.

In another method for measuring leukotriene, which is a mediator, leukotriene is separated from blood, and a calcium ionophore is added to carry out a reaction to release leukotriene from basophils. Then, an ice-cooled acetonitrile / methanol / acetic acid solution containing prostaglandin is added to stop the reaction, left at -20 ° C for 1 hour, centrifuged, and the amount of leukotriene in the reaction solution is measured by HPLC. Have been.

As described above, in the method for measuring leukotriene, which is a mediator released from blood cells, the leukotriene has a very low blood concentration of leukotriene in spite of its absorption characteristic of about 280 nm. Further, since the protein contained in blood also has an absorption around 280 nm, it is impossible to measure the amount of leukotriene in whole blood without separating target cells from blood. Therefore, in order to measure the amount of leukotriene released from blood cells, an operation of separating target cells from blood is required.

As described above, most methods for measuring a mediator released from blood cells require an operation for separating plasma, serum and target cells from blood. This is used when trying to quantify the measurement target because blood contains proteins such as albumin, globulin, antibodies, inorganic substances such as sodium and potassium, and organic substances such as sugars and lipids. Reaction specificity (sensitivity) due to factors such as an increase in the blank value and inhibition of the reaction by blood contaminants in the fluorescence, colorimetric, and luminescent methods that are detection systems
Is caused. As described above, in the measurement of a mediator, a separation operation in several steps is generally required to separate a target mediator from other blood components, which is an extremely complicated and complicated method.

On the other hand, as a method for further simplifying the separation of blood and a desired mediator, Japanese Patent Application Laid-Open No.
A method for releasing histamine from blood cells using whole blood according to 9948 is known. Specifically, 25 μl of heparin-collected blood and 25 μl of the allergen solution were dispensed into a glass plate-immobilized microplate, and
C. for 90 minutes to release histamine from basophils. A method is disclosed in which a microplate on which histamine is adsorbed is washed and then measured by a fluorescence method.
However, in this method, reproducibility is poor due to insufficient separation of blood components from the desired mediator,
Sufficient sensitivity required for diagnosis has not been obtained.

As described above, when a cell reaction is determined with good reproducibility, a target substance and other components must be separated, and when blood is collected from a subject, one blood collection tube is used. Separation operation by centrifuging one and sedimenting blood cells,
Dispensing operations of the plasma and serum obtained by centrifugation into other containers, or the work of separating blood cells from blood using blood cell-specific antibodies are required. The work time was increased, and the cost was increased accordingly.

Further, even if there is a method for simplifying the operation of separating a blood component from a target substance, the reproducibility is poor due to the influence of components in blood, and the results and patient Since a result different from the above-mentioned pathological condition may be obtained, a simple and highly reproducible blood processing method capable of simplifying a step of separating a blood component and a target substance has been desired as a method for determining these cell reactions.

[0012]

If a simple and reproducible blood processing method can be established as a method for determining a cell reaction, accurate measurement values can be obtained, and as a result, the pathology of the subject can be accurately reflected. In addition, there is no need to re-examine abnormal values. In addition, the measurement operation can be simplified, and the cost of a practitioner such as an inspection center can be greatly improved. As described above, improvement in reproducibility and simplification are indispensable requirements in proceeding with inspection in this field. Accordingly, an object of the present invention is to provide a blood processing method and a processing composition thereof, which have high reproducibility and can be simplified in a method for determining a cellular reaction in blood.

[0013]

Means for Solving the Problems The present inventor has conducted intensive studies on the above problems, and as a result, found that a chelating agent,
The present inventors have found that the above problem can be solved by adding an anticoagulant having no chelating ability and a metal salt capable of eluting a divalent metal ion in an aqueous medium, and have accomplished the present invention.

That is, the present invention is characterized in that a chelating agent, an anticoagulant having no chelating ability, and a metal salt capable of eluting a divalent cation in an aqueous medium are added to blood. Provided are a blood treatment method and a treatment composition for a cell reaction. The blood used in the present invention may be blood obtained by diluting whole blood with a physiological salt solution 1 to 10 times, and preferably whole blood.

The chelating agent in the present invention is a substance capable of binding to a divalent metal ion in an aqueous solution, and has a molar concentration ratio of 2 to 1 per molecule of the chelating agent in blood.
It is a substance that causes blood coagulation within 1 hour under room temperature conditions when 0-fold molecular calcium is present. Examples include EDTA, citric acid, oxalic acid, crown ether, nitrilotriacetic acid, and the like, but preferably EDTA-2 contained in a commercially available blood collection tube.
Na and EDTA-2K are preferred, and more preferably,
EDTA-2Na is preferred. The concentration at the time of the blood cell reaction may be 1 to 50 mM, preferably 1.5 to 50 mM.
14 mM, more preferably 2-3.5 mM, is preferred.

The anticoagulant having no chelating ability in the present invention is an anticoagulant that does not cause blood coagulation in the presence or absence of 1 to 10 mM calcium in blood. An anticoagulant that does not coagulate within 1 hour at room temperature in the presence of 2 mM calcium. Examples of anticoagulants include heparin, plasmin, hirudin, antithrombin III,
Thrombomodulin, prostaglandin, Chica
Go blue, Germanin, antibodies to blood coagulation factors, receptors for blood coagulation factors, and the like. Blood coagulation factors include factor VIII and VI
Factor I, Factor IX, Factor X, Factor XI, XII
There is a factor, factor XIII, etc., and any antibody or receptor against it may be used. These anticoagulants preferably include sodium heparin, lithium heparin and the like, and more preferably sodium heparin. The concentration during the blood cell reaction is
1 to 50 U / ml, preferably 5 to 30 U / ml is desirable. Here, 1U is equivalent to 1 unit defined by the Japanese Pharmacopoeia.

In the present invention, the divalent cation is:
Calcium ion (Ca ²⁺ ), magnesium ion (M
g ²⁺ ), manganese ion (Mn ²⁺ ), zinc ion (Zn
²⁺ ), cadmium ion (Cd ²⁺ ), and copper ion (C
u ²⁺ ) and the like, and preferably a calcium ion or a magnesium ion. Examples of metal salts capable of generating divalent cations in an aqueous solution include chlorides, sulfates, carbonates, nitrates, and phosphates. For example, CaCl ₂ , MgCl ₂ , MnCl ₂ , Zn
Cl ₂ , CdCl ₂ , CuCl ₂ , CaSO ₄ , MgS
O ₄ , MnSO ₄ , ZnSO ₄ , CdSO ₄ , CuSO ₄ ,
Ca (NO ₃ ) ₂ , Mg (NO ₃ ) ₂ , Mn (NO ₃ ) ₂ , Z
n (NO ₃ ) ₂ , Cd (NO ₃ ) ₂ , Cu (NO ₃ ) ₂ , Ca
CO ₃ , MnCO ₃ , CdCO ₃ , CaHPO ₄ , MgH
PO ₄ , Zn ₃ (PO ₄ ) _{2 and the} like can be mentioned, but CaCl ₂ and MgCl ₂ are preferable. The concentration at the time of the blood cell reaction is desirably 1 to 200 mM, and the molar concentration ratio with the chelating agent is 1 to 200 times the ratio of the metal salt to 1 molecule of the chelating agent, preferably 1 molecule of the chelating agent. 1 to 50 times is desirable.

The cell reaction used in the present invention is a reaction for expressing a protein on a cell or a reaction for releasing an organic compound such as a protein from a cell, and is given when a cell is stimulated or when it is not stimulated at all. In the state, it may be released in the metabolism of the cell. The term "stimulation of a cell" as used herein means to activate a signal transduction system in a cell by adding a substance from the outside and binding to a substance on the cell or penetrating into the cell. Examples of the signal transduction system include protein phosphorylation and protease activation, and the action of increasing the intracellular calcium concentration by inositol triphosphate. As an example of the case where some stimulus is given to cells, in the release of histamine and leukotriene from basophils, allergen binds to two molecules of allergy-specific IgE antibody bound to FcεRI receptor on basophils, Cross-linking of a specific IgE antibody activates a phosphorylase, which is an intracellular signal transduction system, causing calcium influx into cells and releasing mediators such as histamine.

An example of release in a non-stimulated state is release of interleukin 4 and interleukin 8 from basophils. And the released organic compound includes a mediator released from blood cells. Mediators include histamine, leukotriene, interleukins, and platelet activating f from basophils.
actor (PAF), Major ba from eosinophils
sic protein (MBP), Eosinoph
ile caticic protein (EC
P), Eosinophil-derived n
eurotoxin (EDN), Eosinophil
peroxidase (EPO), Charcot-
beydencrystal (CLC), thromboxane, etc., and histamine, leukotriene, PAF, and interleukin, which are preferably released from basophils, are preferred.

Regarding the method for treating blood, the order of adding the three compositions, namely, a chelating agent, an anticoagulant having no chelating ability, and a metal salt capable of eluting a divalent cation in an aqueous medium is not limited. In any case, the cell reaction can be determined with good reproducibility regardless of the order of addition. For example, at the time of blood collection, only the chelating agent is added,
An anticoagulant having no chelating ability at the time of cell reaction and a metal salt capable of eluting divalent cations in an aqueous medium may be added, or an anticoagulant having no chelating ability at the time of blood collection may be added. In addition, a chelating agent and a metal salt capable of eluting a divalent cation in a water-soluble medium during a cell reaction may be added. In addition, a chelating agent, an anticoagulant having no chelating ability, and a metal salt capable of eluting a divalent cation in an aqueous medium may be added at the time of blood collection, and a cell reaction may be performed using the metal salt.

At this time, the method of adding the chelating agent, the anticoagulant having no chelating ability, and the metal salt capable of eluting divalent cations in an aqueous medium may be either a powder or an appropriate buffer. It may be dissolved therein. Suitable buffers include Pipes buffer, phosphate buffer, acetate buffer, G
Optimum pH for cell stabilization with wood buffer etc.
Is preferably adjusted to a range of 5-1.
It is desirably adjusted to 0, preferably 6 to 9. If necessary, sodium chloride, sodium acetate, potassium chloride, potassium acetate and sodium azide as a preservative may be added.

With respect to the material of the container at the time of addition, polystyrene, polypropylene, Teflon, polyethylene,
Plastics such as methylbenthene resin (TPX), fluororesin, acrylic resin, polycarbonate, polyurethane, and vinyl chloride resin; metals such as stainless steel and titanium;
Anything such as glass and rubber can be used. Regarding the color of the container, any material such as transparent, translucent, and brown can be used.

As a specific method, a chelating agent EDTA is added to blood to which heparin is added as an anticoagulant.
Then, calcium chloride is added as a metal salt, and a suspected allergen in the subject is added to the composition to release a mediator such as histamine from blood cells. Then, the released histamine and the like need only be directly measured by a known measuring method.

The allergen referred to herein is a substance that causes an allergic reaction, and is used in ordinary tests such as an allergen that is commercially available as a reagent for measuring the amount of specific IgE antibody and an allergen that is commercially available as an allergen extract. Any allergens are acceptable. For example, pollen such as cedar and duckweed, food such as milk and egg white, fungi and the like can be mentioned.

In another usage example, EDTA as a chelating agent is used, and heparin as an anticoagulant and a divalent heavy metal as a metal salt are added to blood collected from EDTA. Then, as described above, the allergen may be added to this composition solution to release the mediator from the blood cells, which may be measured by a known measuring method. Further, in another example, blood is collected from a subject and the blood contains the chelating agent E.
DTA, heparin as an anticoagulant, and 2 as a metal salt
As described above, an allergen may be added to this composition solution, a mediator may be released from blood cells, and this may be measured by a known measurement method.

The determination method is a method for quantifying the amount of the organic compound released by the cell reaction, or a method for confirming the amount visually. Methods for quantifying the amount of an organic compound include a method in which a target organic compound is captured on a carrier fixed to a support and measured by a detection system, and a method in which an organic compound is separated by a column and then measured by a detection system. Can be The method of visually confirming is a method of visually confirming the state of blood cells after a cell reaction. Preferably, the determination method is a method of quantifying an organic compound,
More preferably, a method of trapping an organic compound on a carrier fixed to a support is desirable, and more preferably, a method of measuring using an adsorbent specific to the organic compound is desirable.

The term "support" as used herein refers to a container for performing a reaction for releasing a mediator from blood cells, and examples thereof include a microplate, a test tube, and magnetic beads. Regarding the material of the support, polystyrene, polypropylene, Teflon, polyethylene, methylbenthene resin (TP
X), fluorine resin, acrylic resin, polycarbonate,
Polyurethane, plastics such as vinyl chloride resin, metals such as stainless steel and titanium, glass and rubber can be used, and the color of the support is brown,
Anything such as transparent or translucent is acceptable.

Further, the fixed carrier is a substance that specifically adsorbs to an organic compound, and examples thereof include an antibody against the organic compound, a receptor, and glass fiber. The detection system is a method for measuring the amount of an organic compound, which includes a colorimetric method, a fluorescent method, a luminescent method, a radioactive method, and the like, and is measured using an EIA method, an RIA method, and a specifically adsorbing carrier. Is the way.

The EIA method referred to herein is an enzyme immunoassay in which an antibody against a mediator is immobilized on a support and the amount of the mediator is measured by the activity of a labeling enzyme.
For example, as a method for determining an organic compound released by a cell reaction using the EIA method, histamine released from basophils can be measured by a commercially available kit. For example, the following is performed. Heparin-collected blood from the subject is reacted with an allergy-specific antigen solution to release histamine from basophils. The released histamine is reacted with an acylating reagent to acylate histamine. Next, as a method for quantifying histamine,
The amount of enzyme-labeled histamine bound to the anti-acylated histamine antibody was measured by adding acylated histamine and enzyme-labeled histamine to a microplate on which anti-acylated histamine antibody was immobilized, and measuring the labeled enzyme activity. This is a method of obtaining the amount of acylated histamine from the obtained amount.

In another method for measuring histamine, according to Japanese Patent Application Laid-Open No. 6-205695, 10 ml of blood contains HA-H
After adding 40 ml of EPES buffer and diluting the blood, 500 μl was dispensed into a test tube, and 50 μl of magnetic particles on which a monoclonal antibody reacting with basophils was fixed.
And a reaction is carried out at room temperature for 5 minutes to carry out a binding reaction between the monoclonal antibody and basophils. Subsequently, after collecting the beads with a magnet and removing the supernatant, the beads are washed once with a buffer solution. Then, the magnetic particles are transferred to an avidin-immobilized microplate, and reacted with an allergy-specific antigen solution to release histamine from basophils. At that time, a biotin-labeled histamine and an enzyme-labeled antihistamine antibody are simultaneously added to the reaction solution. A method is disclosed in which after washing, the enzyme activity of a biotin-labeled histamine-enzyme-labeled antihistamine antibody complex bound to an avidin-immobilized antibody is determined and the amount of histamine is measured.

Leukotriene, which is another organic compound, can be measured by an EIA method using an anti-leukotriene antibody by a commercially available kit. Interleukin 3, an organic compound released from other blood cells, can be measured by a commercially available kit. For example, the following is performed. Plasma or serum is added to a microplate on which anti-interleukin 3 antibody is immobilized, and a reaction is performed.
To combine. After washing the reaction solution, an enzyme-labeled anti-interleukin-3 antibody was added and the reaction was performed.
And enzyme-labeled anti-interleukin-3. This is a method of measuring the amount of interleukin 3 by measuring the enzyme activity of the labeled antibody. Further, another interleukin, interleukin 6, is disclosed in
According to 271862, serum is added to the microplate on which the anti-interleukin 6 monoclonal antibody is immobilized, and left at room temperature for 2 to 3 hours to allow the immobilized antibody and interleukin 6 to bind. After removing the reaction solution and washing, a biotinylated anti-interleukin-6 polyclonal antibody is added and reacted at 37 ° C. for 1 hour to bind the interleukin-6 and the biotinylated antibody. Next, enzyme-labeled streptavidin was added, and the mixture was allowed to stand at 37 ° C. for 30 minutes to bind the biotinylated antibody and enzyme-labeled streptavidin. After removing the reaction solution, washing was performed three times or more.
There is disclosed a method of adding methylumbelliferyl-β-D-galactoside and measuring the fluorescence intensity of released 4-methylumbelliferone.

The RIA method referred to herein is an immunoassay using a radiolabel. For example, as a method for determining an organic compound released by a cell reaction using the RIA method, in the measurement of histamine released from basophils (clinical test vol. 26 no. 2 1982/2), for example, To do. After blood collection under the conditions of EDTA addition, plasma is separated by centrifugation. Plasma or standard histamine and an acylation buffer are added to an acylating reagent tube to acylate histamine in plasma. Next, ¹²⁵ I-labeled histamine is added and reacted in an antihistamine antibody-fixed tube. A method has been reported in which the reaction solution is removed after the reaction, and the amount of histamine in plasma is determined by measuring the radioactivity bound to the antihistamine antibody tube.

In the measurement of leukotriene, which is another organic compound, a sample or a standard unlabeled leukotriene, an anti-leukotriene antibody, and ³ H-labeled leukotriene are reacted at 4 ° C. for 5 to 24 hours, and then dextran charcoal and ice. Incubate for 10 minutes. After centrifugation, the supernatant is collected and the amount of leukotriene is determined by measuring radioactivity.

Measurement of PAF which is another organic compound
Is a sample or standard unlabeled PAF, anti-PAF antibody,
¹²⁵Reaction of I-labeled PAF and secondary antibody at room temperature for 5 to 24 hours
After the reaction, the radioactivity of the precipitate obtained by centrifugation is measured.
To determine the amount of PAF. Free from eosinophils
For information on how to measure ECP,
The kits on sale are as follows. Anticoagulant
Blood is collected in the presence and separated into plasma or serum by centrifugation
Take 50 μl each of the standard ECP and the sample into a test tube,
¹²⁵50 I-labeled human ECP and rabbit anti-human ECP antibody
Add in aliquots and incubate at room temperature overnight. to this
Add 2 ml of sheep anti-rabbit IgG antibody and add at room temperature for 30 minutes.
After incubation, remove the supernatant by centrifugation, and
Method of measuring ECP amount by measuring Unter
It has been known.

The method of measuring an organic compound by a detection system after separating the organic compound by a column is to utilize the physical properties of the mediator, such as ionicity, hydrophobicity, affinity, etc., and to carry out the mediator by a resin-filled column. This is a separation method, and examples include an HPLC method. HPLC
As a method for determining an organic compound using the method, there is, for example, a method for measuring histamine according to JP-A-6-205695. For example, after histamine is separated by passing a sample through a column filled with a cation exchange resin and histamine is added thereto, 0.1% orthophthalaldehyde and NaOH are added, and a histamine-orthophthalaldehyde binding reaction is performed as described below, and histamine is fluoresced. A method has been reported in which HCl is added as a body, the reaction is stopped, and the amount of histamine released is measured by measuring the fluorescence intensity. In the measurement of leukotriene, another organic compound, leukocytes are separated from blood, calcium ionophore is added to release leukotriene from basophils, and the reaction is performed with an ice-cold acetonitrile / methanol / acetic acid solution containing prostaglandin. Was stopped and left at −20 ° C. for 1 hour, followed by centrifugation and separation of leukotriene by reverse layer chromatography to measure the absorption at 280 nm, which is the absorption characteristic of leukotriene.
This is a method for measuring the amount of leukotriene.

The carrier specifically adsorbing the organic compound mentioned here includes glass fiber, antibody, enzyme, receptor, lectin and the like. As a method for measuring using a carrier that specifically adsorbs an organic compound, Japanese Patent Application Laid-Open No.
In the measurement of histamine released from blood cells, heparin-collected blood and allergen are dispensed into a microplate on which glass fibers are immobilized, histamine is released from basophils, and histamine bound to glass fibers. A method for measuring the amount by a fluorescence method is disclosed.

[0037]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[0038]

Example 1 Histamine released from blood cells was measured using one healthy volunteer blood sample. At the time of blood collection, 25 μl of blood to which 5 mL of blood was added with sodium EDTA (manufactured by Dojindo Chemical Co., Ltd.) so that the EDTA sodium concentration was 1 mM during the histamine release reaction, and 50 mM calcium chloride (Wako Pure Chemical Industries, Ltd.) ), 70 mM sodium acetate (manufactured by Wako Pure Chemical Industries), 2.5 mM potassium acetate (manufactured by Wako Pure Chemical Industries), 10 U / ml sodium heparin (Hoechst Marion Roussel) and 0.005% A microplate in which 25 μl of a human anti-IgE antibody solution (manufactured by DAKO) prepared with Pipes buffer (manufactured by Dojindo) (pH 7.4) containing sodium chloride (manufactured by Wako Pure Chemical Industries) is immobilized on a glass fiber. And histamine was released from basophils. After the reaction, the microplate was washed, and a surfactant was added for 200 hours.
Add μl and react at 37 ° C. for 60 minutes to wash out impurities in blood attached to the glass fiber. Thereafter, a 0.05% orthophthalaldehyde solution (manufactured by Wako Pure Chemical Industries, Ltd.) prepared with sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was used for 75 times.
After dispensing μl to form a phosphor, the reaction was stopped by dispensing 75 μl of 0.6% perchloric acid (manufactured by Wako Pure Chemical Industries, Ltd.), the fluorescence intensity was measured, and the amount of histamine was determined from the histamine standard solution. I asked. The test was performed 5 times, and the value obtained by dividing the standard deviation by the average value was multiplied by 100 to obtain the Coefficient of V.
Arrangement (CV) (%) was determined. As a result C
The V value was 7.8%.

[0039]

Example 2 A test was performed in the same manner as in Example 1 except that the sodium EDTA concentration was 1, 25, and 50 mM during the histamine release reaction. As a result, the CV value is 9%,
8.5% and 9.7%.

[0040]

Comparative Example 1 Using one healthy volunteer blood, histamine released from blood cells was measured. For 5 ml of blood at the time of blood collection, 25 μl of blood to which heparin sodium was added so that the concentration of heparin sodium was 10 U / ml during the histamine release reaction, and 50 mM calcium chloride, 70 mM sodium acetate, 2.5 mM acetate Potassium and 0.00
Pipes buffer containing 5% sodium azide (pH
Histamine was measured in the same manner as in Example 1 using 25 μl of the human anti-IgE antibody solution prepared in 7.4). As a result, the CV value was 123%. When the sodium EDTA concentration was in the range of 1 to 50 mM, the CV value was reduced to 7.8 to 9% in combination with the result of Example 1. As is apparent from the result that the CV value indicating the degree of the variation significantly decreased, the reproducibility of the cell reaction was improved by the addition of the sodium EDTA concentration of 1 to 50 mM.

[0041]

Example 3 A test was conducted in the same manner as in Example 1 except that the calcium chloride concentration was 1, 100, and 200 mM during the histamine release reaction. As a result, the CV value was 8.5.
%, 8.5% and 8.3%.

[0042]

Comparative Example 2 A test was performed in the same manner as in Example 1 except that the calcium chloride concentration was 0 mM during the histamine release reaction. As a result, the CV value was 150%. When the calcium concentration was in the range of 1 to 200 mM, the CV value was reduced to 7.8 to 8.5% in combination with the result of Example 1.
As is clear from the results, the calcium concentration was 1 to 20.
Within the range of 0 mM, the reproducibility of the cell reaction was improved.

[0043]

Example 4 A test was conducted in the same manner as in Example 1, except that the sodium heparin concentration was 1, 20, and 50 U / ml during the histamine release reaction. As a result, the CV value is
8.3%, 8.0% and 8.4%.

[0044]

Comparative Example 3 A test was conducted in the same manner as in Example 1, except that the concentration of heparin sodium was 0 mM during the histamine release reaction. As a result, the CV value is 143%,
In the range of heparin sodium concentration of 1 to 50 U / ml,
Together with the results of Example 1, the CV value was reduced to 7.8 to 8.4%. As is clear from the results, the reproducibility of the cell reaction was improved in the range of the heparin concentration of 1 to 50 U / ml.

[0045]

Example 5 A test was performed in the same manner as in Example 1 except that sodium EDTA was sodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the CV value was 8.2%.

[0046]

Example 6 A test was conducted in the same manner as in Example 1 except that sodium EDTA was sodium oxalate (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the CV value was 8.1%. From the results of Example 1, Example 5, Example 6, and Comparative Example 1, even when the chelating agent was sodium EDTA, sodium citrate, or sodium oxalate, the CV value was 7.8 to 8.2%. And the reproducibility of the cell response was improved.

[0047]

Example 7 A test was performed in the same manner as in Example 1 except that sodium heparin was plasmin (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the CV value was 8.9%.

[0048]

Example 8 A test was performed in the same manner as in Example 1 except that the heparin sodium was thrombin (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the CV value was 8.6%.
From the results of Example 1, Example 7, Example 8, and Comparative Example 2,
Even if the anticoagulant having no chelating ability was heparin sodium, plasmin, or thrombin, the CV value was reduced to 7.8 to 8.9%, and the reproducibility of the cell reaction was improved.

[0049]

Example 9 A test was performed in the same manner as in Example 1 except that calcium chloride was magnesium chloride (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the CV value was 9.7%.

[0050]

Example 10 A test was conducted in the same manner as in Example 1 except that calcium chloride was manganese chloride (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the CV value was 9.9%.
From the results of Example 1, Example 9, Example 10, and Comparative Example 3, even when the divalent cation is a calcium ion, a magnesium ion, or a manganese ion, the CV value is 7.8 or more.
This decreased to 9.9%, and the reproducibility of the cell reaction was improved.

[0051]

Example 11 A test was performed in the same manner as in Example 1 except that calcium chloride was calcium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the CV value was 9.3%.

[0052]

Example 12 A test was performed in the same manner as in Example 1 except that calcium chloride was calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the CV value was 9.5%. From the results of Example 1, Example 11, Example 12, and Comparative Example 3, even if the metal salt is chloride, sulfate, or carbonate,
The CV value was reduced to 7.8 to 9.5%, and the reproducibility of the cell reaction was improved.

[0053]

Example 13 Using one healthy volunteer blood sample,
Histamine released from blood cells was measured. 5 ml of blood at the time of blood collection and EDTA at the time of histamine release reaction
Sodium concentration 1 mM, heparin sodium concentration 1
The concentration at the time of histamine release reaction with 25 μl of blood added with sodium EDTA and sodium heparin so as to be 0 U / ml was 50 mM calcium chloride, 70 mM
M sodium acetate, 2.5 mM potassium acetate, and
Human anti-IgE antibody solution 25 prepared with Pipes buffer (pH 7.4) containing 0.005% sodium azide
Histamine was measured in the same manner as in Example 1 using μl. As a result, the CV value was 8.0%, and a result equivalent to the CV value of Example 1 of 7.8% was obtained. As is clear from these results, the reproducibility of the cell reaction was improved even when a chelating agent and an anticoagulant having no chelating ability were simultaneously added to blood.

[0054]

Example 14 Using one healthy volunteer blood sample,
Histamine released from blood cells was measured. For blood 5 ml at the time of blood collection, 25 μl of blood to which heparin sodium was added so that the concentration of heparin sodium became 10 U / ml at the time of histamine release reaction, and 50 mM calcium chloride, 70 mM sodium acetate, 2.5 mM acetate Potassium, 1 mM ED
Histamine was measured in the same manner as in Example 1 using 25 μl of a human anti-IgE antibody solution adjusted with Pipes buffer (pH 7.4) containing sodium TA and 0.005% sodium azide. As a result, CV
(%) Was 8.0%, and a result equivalent to the CV value of Example 1 of 7.8% was obtained. As evident from this result,
Even when an anticoagulant having no chelating ability was added to blood and a chelating agent and a metal salt were added to the buffer, the reproducibility of the cell reaction was improved.

[0055]

Example 15 Using one example of healthy volunteer blood,
Histamine released from blood cells was measured. At the time of blood collection, EDT was adjusted so that the EDTA sodium concentration was 1, 25, and 50 mM during the histamine release reaction for 5 ml of blood.
A concentration of 50 mM calcium chloride, 70 mM
M sodium acetate, 2.5 mM potassium acetate, 10 U /
50 μl of a human anti-IgE solution adjusted with a Pipes buffer (pH 7.4) containing 0.1 ml of sodium heparin and 0.005% sodium azide was dispensed into a plastic tube, reacted at 37 ° C. for 90 minutes, and subjected to basophils. To release histamine. Thereafter, centrifugation is performed to remove hemocytes, and then the released histamine is reacted with an acylating reagent to acylate histamine. 200 μl of acylated histamine and enzyme-labeled histamine were added to a microplate on which an anti-acylated histamine antibody was immobilized, and reacted at 5 ° C. for 18 hours. After washing, 200 μl of enzyme substrate was added and reacted at 25 ° C. After one minute, stop reaction liquid 50 μl
Was added, and the absorbance at 405 nm was measured. As a result, C
The V values were 9.2%, 8.8%, and 9.5%.

[0056]

Comparative Example 4 Using one healthy volunteer blood, histamine released from blood cells was measured. At the time of blood collection, 50 ml of blood to which 5 ml of blood was added with heparin sodium so that the concentration of sodium heparin became 10 U / ml during the histamine release reaction was 50 mM calcium chloride, 70 mM sodium acetate, and 2.5 mM acetate. Potassium and 0.00
Pipes buffer containing 5% sodium azide (pH
Histamine was measured in the same manner as in Example 15 using 50 μl of the human anti-IgE antibody solution prepared in 7.4). As a result, the CV value was 140%, and the EDTA
When the sodium concentration is in the range of 1 to 50 mM, the CV value is 8.
It decreased to 8 to 9.5%. As is apparent from the results, the reproducibility of the cell reaction was improved in the range of the sodium EDTA concentration of 1 to 50 mM.

[0057]

Example 16 Using one example of healthy volunteer blood,
Histamine released from blood cells was measured. At the time of blood collection, EDT was adjusted so that the EDTA sodium concentration was 1, 25, and 50 mM during the histamine release reaction for 5 ml of blood.
50 μl of each of three types of blood to which sodium A was added
And 50 mM calcium chloride, 70 mM sodium acetate, 2.5 mM potassium acetate, 10 U / ml sodium heparin, and 0.0
Pipes buffer containing 0.05% sodium azide (pH
25 μl of the human anti-IgE solution prepared in 7.4) is dispensed into a tube and reacted at 37 ° C. for 90 minutes to release histamine from basophils. Thereafter, centrifugation is performed to remove blood cells, and then 500 μl of released histamine, μl of standard histamine, and 50 μl of acylation buffer are added to the acylating reagent tube to acylate the released histamine. Next, 1 ml of ¹²⁵ I-labeled histamine is added and reacted four times in an antihistamine antibody-fixed tube for 16 hours.
After the reaction, the reaction solution was removed, and the radioactivity bound to the antihistamine antibody tube was measured. As a result, the CV value becomes 9.
3%, 9.0%, and 9.5%.

[0058]

Comparative Example 5 Histamine released from blood cells was measured using one healthy volunteer blood sample. For 5 ml of blood at the time of blood collection, 25 μl of blood to which heparin sodium was added so that the concentration of heparin sodium was 10 U / ml during the histamine release reaction, and 50 mM calcium chloride, 70 mM sodium acetate, 2.5 mM acetate Potassium and 0.00
Pipes buffer containing 5% sodium azide (pH
Histamine was measured in the same manner as in Example 16 using 25 μl of the human anti-IgE antibody solution prepared in 7.4). As a result, the CV value was 140%, and the EDTA
When the sodium concentration is in the range of 1 to 50 mM, the CV value is 9 to
It decreased to 9.5%. As is apparent from this result, E
In the range of sodium DTA concentration of 1 to 50 mM,
The reproducibility of the cell reaction was improved.

[0059]

Example 17 Using one example of healthy volunteer blood,
Leukotriene released from blood cells was measured. At the time of blood collection, 5 μl of blood was added to each of 25 μl of three kinds of blood to which sodium EDTA was added so that the concentration of sodium EDTA became 1, 25, and 50 mM at the time of leukotriene release reaction.
1. 0 mM calcium chloride, 70 mM sodium acetate,
5 mM potassium acetate, 10 U / ml heparin and 0.
Pipes buffer containing 005% sodium azide (p
25 μl of the human anti-IgE solution prepared in H7.4) was dispensed into a tube, and reacted at 37 ° C. for 90 minutes to release leukotriene from basophils. Then, after centrifugation to remove blood cells, the reaction was stopped with an ice-cold acetonitrile / methanol / acetic acid solution containing prostaglandin,
After standing at -20 ° C for 1 hour, leukotriene was separated by centrifugation and reverse-layer chromatography, and the absorption at 280 nm, which is the absorption characteristic of leukotriene, was measured to measure the amount of leukotriene. As a result, the CV values are:
They were 8.1%, 8.8% and 9.3%.

[0060]

Comparative Example 6 Leukotriene released from blood cells was measured using one healthy volunteer blood sample. For blood 5 ml at the time of blood collection, 25 μl of blood to which heparin sodium was added so that the heparin sodium concentration was 10 U / ml at the time of histamine release reaction, and 50 mM calcium chloride, 70 μm as a concentration at the time of leukotriene release reaction.
mM sodium acetate, 2.5 mM potassium acetate, and
Human anti-IgE antibody solution 25 prepared with Pipes buffer (pH 7.4) containing 0.005% sodium azide
Leukotriene was measured in the same manner as in Example 17 using μl. As a result, the CV value was 120%, and in the range of sodium EDTA concentration of 1 to 50 mM,
Together with the results of Example 17, the CV value was 8.1 to 9.3%.
And declined. As is apparent from the results, the reproducibility of the cell reaction was improved in the range of the sodium EDTA concentration of 1 to 50 mM.

[0061]

The present invention relates to a method for determining a blood cell reaction, comprising a blood, a chelating agent, an anticoagulant having no chelating ability, and a metal salt capable of eluting a divalent cation in an aqueous medium. This shows that cell function can be measured with good reproducibility without blood separation by blood treatment using the composition combined from the group. Therefore, the effects of the present invention are expected to be able to enhance the reproducibility of the cell function test, to reduce the work time of the operator by simplifying the operation, and to reduce the cost associated therewith.

Claims (20)

[Claims] 1. A treatment of blood subjected to a cell reaction, comprising adding a chelating agent, an anticoagulant having no chelating ability, and a metal salt capable of eluting a divalent cation in an aqueous medium. Method. 2. The treatment method according to claim 1, wherein the cell reaction is a mediator release reaction from blood cells. 3. The method according to claim 1, wherein the chelating agent is Ethylendiam.
ine tetraacetic acid (EDT
The processing method according to claim 1, comprising one or more selected from the group consisting of A), citric acid, and oxalic acid. 4. An anticoagulant having no chelating ability,
4. A composition comprising at least one member selected from the group consisting of heparin, plasmin, protease, azo dye, hirudin, dicoumarol, thrombomodulin, an antibody against a blood coagulation factor, and a receptor that binds to a blood coagulation factor.
The processing method described. 5. The treatment method according to claim 1, wherein the divalent cation includes at least one selected from the group consisting of calcium, magnesium, manganese, zinc, cadmium, and copper. 6. The treatment method according to claim 1, wherein the metal salt contains at least one selected from the group consisting of chlorides, sulfates, carbonates, nitrates, and phosphates. 7. The mediator may be histamine, leukotriene, platelet activating.
The method according to claim 2, wherein the treatment method is a factor (PAF) or a cytokine. 8. The concentration of the chelating agent may be 1 to 3 during the cell reaction.
50 mM, and the molar concentration ratio with the metal salt is 1
The processing method according to any one of claims 1 to 7, wherein the metal salt is 1 to 200 with respect to the amount of the metal salt. 9. The anticoagulant having no chelating ability has a concentration of 1 to 50 U / ml during a cell reaction.
The processing method described. 10. The concentration of a metal salt capable of eluting a divalent cation is 1 to 200 mM during a cell reaction.
The processing method described. 11. A composition for treating blood to be subjected to a cell reaction, comprising a chelating agent, an anticoagulant having no chelating ability, and a metal salt capable of eluting a divalent cation in an aqueous medium. 12. The composition according to claim 11, wherein the cellular reaction is a mediator release reaction from blood cells. 13. The chelating agent may be Ethylendia.
mine tetra acidic acid (ED
The composition according to claim 11, comprising at least one selected from TA), citric acid, and oxalic acid. 14. An anticoagulant having no chelating ability includes heparin, plasmin, protease, azo dye,
2. The composition according to claim 1, which comprises at least one member selected from the group consisting of hirudin, dicoumarol, thrombomodulin, an antibody against a blood coagulation factor, and a receptor that binds to a blood coagulation factor.
14. The composition according to 1 to 13. 15. The composition according to claim 11, wherein the divalent cation includes at least one selected from the group consisting of calcium, magnesium, manganese, zinc, cadmium, and copper. 16. The metal salt is chloride, sulfate, carbonate,
The composition according to any one of claims 11 to 15, comprising at least one member selected from the group consisting of nitrates and phosphates. 17. The mediator may be histamine, leukotriene, platelet activating.
17. The composition according to claim 12, which is a factor (PAF) or a cytokine. 18. The concentration of the chelating agent may be 1 during cell reaction.
The composition according to any one of claims 11 to 17, wherein the metal salt is 1 to 200 at a molar concentration ratio to the metal salt of 1 to 200 mM. 19. The concentration of an anticoagulant having no chelating ability is 1 to 50 U / ml during a cell reaction.
The composition of any one of claims 18 to 18. 20. The concentration of a metal salt containing a divalent cation is:
20. The composition according to claims 11 to 19, which is 1 to 200 mM during a cell reaction.