ES2354201T3 - CONTAINERS AND KITS FOR THE DETERMINATION OF CELLULAR FUNCTIONS AND METHODS FOR THE DETERMINATION OF THE SAME. - Google Patents

Abstract

THE INVENTION REFERS TO CONTAINERS FOR THE DETERMINATION OF CELLULAR FUNCTIONS THAT ARE EASY TO OPERATE AND ARE LESS DANGEROUS, ALLOWING A HIGH PRECISION DETERMINATION. SPECIFICALLY, A CONTAINER FOR THE DETERMINATION OF CELLULAR FUNCTIONS TO BE USED WITH THE PHYSIOLOGICALLY ACTIVE SUBSTANCES OF ANALYSIS PRODUCED BY THE HEMATOCITES, CONSTITUTED SO THAT THE QUANTITY OF INDUCTIVE MATERIALS OF THE PRODUCTION OF THE ACTIVITY OF THE PRODUCTS, LEVEL THAT DOES NOT CAUSE THE HEMATOCITS TO PRODUCE THE SUBSTANCES, AS IT IS DETERMINED EXTRACTING WATER FROM THE CONTAINER, IN AN AMOUNT EQUAL TO THAT OF A BLOOD SAMPLE TO BE ANALYZED; AND ANOTHER CONTAINER FOR THE SAME, WHERE THE MATERIALS THAT INDUCE THE PRODUCTION OF SUBSTANCES, WHEN THEY BECOME CONTACT WITH THE BLOOD, ARE PLACED IN A STATE ABLE TO CONTACT THE BLOOD, DECREASING THE AMOUNT OF THE MATERIALS IN THE CONTAINER SO THAT IT DOES NOT AFFECT THE ANALYSIS OF THE SUBSTANCES.

Description

Field of the Invention

 The present invention relates to a kit for the measurement of cellular functions involved in immune and inflammatory reactions, and more particularly to a kit for the measurement of cellular functions that is performed by determining cytokines as physiologically active substances, produced by cells. blood cells including granulocytes, monocytes, macrophages, lymphocytes or the like.

Description of the prior art

 Leukocytes, such as granulocytes, monocytes, macrophages and 10 lymphocytes, play various roles in diversified bioprotective reactions, including immune and inflammatory reactions in blood or respective organs. It is known that these cells show important functions in various morbidities including infectious diseases; inflammatory diseases such as hepatitis and nephritis; 15 allergic immune diseases such as rheumatoid arthritis and asthma; and cancer, and the functions of these cells are suppressed or enhanced as morbidity varies.

 It is also known that various drugs, such as anti-inflammatory drugs, immunosuppressants, immunopotentiators and anticancer drugs, are useful in the therapy of these diseases, in which the functions of these cells are simultaneously suppressed or enhanced. It is important, therefore, to examine the functions of these cells, whereby the morbidities of various diseases, effects and side effects of drugs can be identified to determine therapeutic schedules, doses of the drugs and the timing of drug administration.

 In view of the reasons described above, a phagocytic granulocyte activity test, a granulocyte bactericidal activity test (active oxygen production capacity), a lymphocyte transformation assay and the like have been conventionally performed in rooms or 30 centers of hospital recognition to measure these cellular functions. Also in recent years, a surface antigen assay has been performed using a flow cytometer and fluorescently labeled monoclonal antibodies against respective surface antigens of various immunocompetent cells. However, the test methods 35

Conventional techniques have required specialized techniques such as cell separation and culture, microscopic measurement or the like, to therefore require time-consuming measurements, IR facilities and expensive equipment.

 In addition, blood monocytes, as well as macrophages in which the 5 monocytes moved to differentiated and mature tissues, have a broad spectrum of functions, for example, such as coming into play in the exclusion of foreign bodies through phagocytosis and immune formation by antigen presentation, or as a secretion of various physiologically active substances, such as cytokine and prostaglandin, to thereby regulate an inflammatory or immune reaction. Like granulocytes and lymphocytes, these monocytes and macrophages also play important roles in various morbidities. It is very important, therefore, to identify the functions of these cells. Particularly in infectious diseases, unlike granulocytes and lymphocytes, monocytes and macrophages show slight changes in terms of cell numbers and mainly amplify their functions, so that the measurement of changes in cellular functions becomes more important ( "Macrophages" document, written by Tohru Tokunaga, Kodansha Scientific, 1st Ed. Published in 1986).

 The tumor necrosis factor α (hereinafter referred to as TNFα), interleukin-1β (hereinafter referred to as IL-1β), and interleukin-6 (hereinafter referred to as IL -6), called all monokine, are cytokines that are produced primarily by leukocytes including monocytes and macrophages, between blood cells, and that come into play in various inflammatory and immune reactions.

 Various methods described to date examine the functions of cytokine production described above from blood or leukocytes separated from blood. For example, in Open Patent Bulletins for Public Inspection No. Hei 2-196961 and Hei 3-285692, 30 methods are described that make lipopolysaccharide (LPS) or blood lectin react to induce the production of cytokines, such as TNFα or IL-1β, which are subsequently quantitatively determined. In addition, methods that make the blood react with polymeric materials having a surface roughness or specific chemical structures to induce the production of TNFα are described in Patent Patent Bulletins Open to Public Inspection No. Hei 6-209992 and Hei 7-67955. . In addition, bioreaction assays that react to blood with polymeric materials that have a specific surface roughness to determine the amount of TNFα or IL are described in the Open Patent Bulletins for Public Inspection No. Hei 7-299732 and Hei 7-151752. -1β 5 produced. In addition, in a Tokkohyo bulletin No. Hei 7-500905, a method is described that measures the immunoactivity of a tested substance by determining the production of cytokines, such as TNFα or IL-1β, induced from human peripheral blood leukocytes.

 However, the methods described above for the measurement of 10 cellular functions that have been conventionally performed in hospital rooms or centers of recognition, as well as the methods described in the bulletins of the patents opened for public inspection mentioned above, present the following problems. That is, all these tests require specialized operations, such as a blood collection operation of a human being examined using an injector and then manually transferring the blood to various reactors such as by pipetting, cell separation to separate the leukocytes and the others, cell culture in order to measure cell functions or the like. This carries the risk that the person performing the test 20 will contract various infectious diseases, such as hepatitis and AIDS, when the person comes into contact with the blood. In addition, there is a possibility that various bacteria or dusts accidentally incorporated into a blood sample during such operations. There is another risk of negatively influencing the measurement results when those contaminants or operations physically stimulate the cells in the blood without need.

 Particularly in conventional methods that employ a specific reactor to collect the blood inside and determine the production of cytokines, specifically of TNFα or IL-1β induced from leukocytes, there has been an occasion in which endotoxin, such as LPS obtained from 30 gram-negative bacteria, it has originally been incorporated into a blood collection equipment, such as an injector, or into a reactor. Since even a very small amount of endotoxin can induce the production of TNFα or IL-1β from leukocytes, it was impossible to obtain reliable measurement results when, for example, the entry of a small amount of powders during a manufacturing process or contamination through the cleaning water employed resulted in the incorporation of a small amount of endotoxin in the blood collection equipment or reactor described above.

 In view of the problems described above, a method 5 is sought for the measurement of cellular functions whose operations are simpler, less risky and more precise than conventional methods.

 In another aspect, anticoagulants have been conventionally used when various physiologically active substances in blood, blood cell functions, blood cell surface antigens or the like are measured.

 However, there is no general pattern for the content of endotoxin in anticoagulant, apart from a guideline given in the document “Endotoxin testing method” in the vademecum of the 13th revised Japanese Pharmacopoeia, which, for anticoagulants used as a drug For injection, it officially establishes 5 EU / kg as a standard for a minimum pyrogen dose specification to a rabbit.

 Endotoxin is a lipopolysaccharide that constitutes a membrane external to the cell wall of gram-negative bacteria, and a very light amount of it is enough to stimulate blood cells, such as leukocytes, to produce physiologically active substances such as various cytokines. including TNFα, IL-1β, IL-6, or granulocyte-macrophage colony stimulating factors. These show various physiological actions such as pyrogenic activity and endotoxin-induced shock (Nippon Igaku-kan "Inflammation and cytokines '87 Inflammation Seminar", 25 pp. 103-108).

 In addition, physiologically active substances, such as the cytokines described above produced from blood cells, interact with each other in an autocrine or paracrine manner to cause additional production of histamines, arachidonate metabolites or various cytokines, 30 to modify various cell functions. blood, and to cause quantitative and qualitative changes of blood cell surface antigens.

 Therefore, if the anticoagulants are contaminated with endotoxin, and if the content of said endotoxin is at a level sufficient to cause the production of physiologically active substances, it would become impossible to make precise measurements of various physiologically active substances in blood, or functions of blood cells and blood cell surface antigens.

 WO 94/26935 refers to a method and a device 5 for detecting and measuring the amount of a cell-associated molecule in a sample of biological fluid, with which a large number of molecules associated with cells comprising cytokines can be detected. .

 Taktak YS et al, "Assay of pyrogens by interleukin-6 release from monocytic cell lines," Journal of Pharmacy and Pharmacology, Vol. 10 43, No. 9, 1991, pages 578-582 describes a method of determining pyrogens by the release of interleukin-6 in pharmaceuticals.

Summary of the invention

 It is an object of the present invention to provide a kit for the measurement of cellular functions, which can eliminate the problems imposed in conventional methods for the measurement of cellular functions, which are of operations simpler and less risky than conventional ones, and that can measure cellular functions with greater precision than conventional ones. twenty

 According to the invention, to fulfill the object described above, a kit is provided as defined in claim 1. The kit comprises a container for the measurement of cellular functions, for use in the determination of physiologically active substances produced at from blood cells, in which an amount of material capable of inducing the production of the physiologically active substances described above, when extracted by collecting water of a volume equal to a volume of liquid to be subjected to the measurement, It is at an insufficient level to induce the production of physiologically active substances from blood cells. 30

 Since, in this vessel for the measurement of cellular functions, as described above, the amount of material capable of inducing the production of physiologically active substances, when extracted by collecting water of a volume equal to a volume of liquid that it will be subjected to measurement, it is limited to an insufficient level to induce the production of physiologically active substances from blood cells, that is, the container for the measurement of cellular functions contains in itself the limited amount specified above of the material capable of inducing the production of physiologically active substances, the collected blood is hardly subjected to unnecessary stimulation during a period 5 from the collection to the measurement, so that a long-term preservation thereof is allowed. This allows the precise measurement of the physiologically active substances present in the collected blood and allows the use of the container in the examination in a precise way of morbidities of patients who have various diseases. 10

 This first container for the measurement of cellular functions is used to obtain control values. It is used in the kit in combination with a second container for the measurement of cellular functions as described below.

 In the first container for the measurement of cellular functions according to the invention of the present application, the material capable of inducing the production of the physiologically active substances described above is an endotoxin, and its content in the container for the measurement of Cellular functions before use is specified not to exceed 0.5 EU / ml as a concentration in an extracted liquid when it is extracted by collecting water of a volume equal to a volume of liquid to be measured.

 In the second container for the measurement of cellular functions, a material that induces the production of physiologically active substances in blood after contact with blood, is housed inside such that it can come into contact with blood, and that the quantity, present in the container before use, of the material capable of inducing the production of physiologically active substances described above is limited so as not to negatively influence the measured values of the physiologically active substances as described above. 30

 In the second container for the measurement of cellular functions according to the invention, although the material capable of inducing the production of physiologically active substances in blood is housed in such a way that it can come into contact with the blood, the content of the material capable of inducing the production of the physiologically active substances, originally present in the container before it is housed, is preferably limited, as described above, so as not to influence the measured values of the physiologically active substances. Therefore, the production of physiologically active substances can be determined very precisely when blood is introduced and placed in contact with the material capable of inducing the production of physiologically active substances to thereby produce physiologically active substances. .

 In the second container for the measurement of cellular functions according to the invention, the material that induces the production of the 10 physiologically active substances described above is endotoxin, and an endotoxin concentration in a resulting complete liquid when contacted with the blood is limited, being in the range of 0.6-1000 EU / ml.

 In addition, anticoagulants are also incorporated into both containers for measuring cell functions according to the invention to prevent blood clotting.

 In addition, the amount of the material capable of inducing the production of physiologically active substances contained in the anticoagulant described above is preferably at an insufficient level to induce the production of physiologically active substances from blood cells when mixed with blood.

 In the present invention, the material capable of inducing the production of physiologically active substances is endotoxin, while the physiologically active substances are cytokines. 25

 At least one species selected from tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) and interleukin-6 (IL-6) can be mentioned as the cytokines described above.

 In addition, an interior of the container for measuring cellular functions, according to the invention, is preferably vacuum. 30

 Furthermore, the containers for the measurement of cellular functions, according to the invention, are combined with a reagent capable of quantifying the physiologically induced substances induced to thereby constitute a kit for the measurement of cellular functions according to claim. 1. An enzyme immunoassay reagent is used as the reagent capable of quantifying induced physiologically active substances.

 The invention of the present application is a kit for the measurement of cellular functions. As mentioned, the kit has a first container for the measurement of cellular functions in which an amount of a material capable of inducing the production of the physiologically active substances described above, when extracted by collecting water that does not contain the material capable of inducing the production of physiologically active substances and has a volume such that it is equal to a volume of liquid that will be subjected to measurement, it results in an insufficient level to induce the production of physiologically active substances from 10 blood cells, and in which anticoagulant content is contained; a second container for the measurement of cellular functions in which a material capable of inducing the production of physiologically active substances in blood when it comes into contact with blood, as well as anticoagulant, are housed in such a way that they can come into contact with the blood, and in which the amount, originally present in the container prior to the housing thereof, of the material capable of inducing the production of physiologically active substances described above is preferably limited so as not to negatively influence the measured values of the substances physiologically active as described above; and a reagent to quantitatively determine the physiologically active substances. That is, the kit for the measurement of cellular functions according to the invention has a constitution that combines the first vessel for the measurement of cellular functions, the second vessel for the measurement of cellular functions, and the reagent for quantification defined above.

 In the kit for measuring cell functions according to the invention, the quantification reagent specified above includes a first enzyme immunoassay reagent for use in determining the amount of physiologically active substances in the blood collected in the first container for the measurement of cellular functions; and a second enzyme immunoassay reagent that is used to determine the amount of physiologically active substances produced by a reaction of the material capable of inducing the production of physiologically active substances with the blood collected in the second container for the measurement of cellular functions , and which is different in sensitivity to the physiologically active substances of the first enzyme immunoassay reagent, as defined in claim 1.

 In addition, in the kit for the measurement of cellular functions according to the invention, the material that induces the production of the physiologically active substances described above is endotoxin, and an endotoxin concentration in a resulting complete liquid when contacted with blood it translates into the range of 0.6 - 100,000 EU / ml.

 The present invention will be explained in detail below.

(First container for measuring cell functions) 10

 For the first container for the measurement of cellular functions, it is required that the amount of material capable of inducing the production of the physiologically active substances described above, when extracted by collecting water devoid of the material capable of inducing the production of physiologically active substances and of a volume equal to a volume of liquid to be measured, is at an insufficient level to induce the production of physiologically active substances from blood cells.

 The extraction method is actually carried out by collecting, in the previous container for the measurement of cellular functions, water 20 devoid of the material capable of inducing the production of physiologically active substances and of a volume equal to a volume of liquid to be subjected to measurement , and stirring with stirring for one hour at 37 ° C.

 The above physiologically active substances are cytokines and the material capable of inducing the production of physiologically active substances is an endotoxin.

 The endotoxin content in an extracted solution resulting from the extraction described above is specified not to exceed 0.5 EU / ml (international endotoxin unit). If the content of the previous endotoxin exceeds 0.5 EU / ml, it is likely that the endotoxin causes a marked induction of cytokines, in the collected blood, and the induced cytokines possibly stimulate various immunocompetent cells to cause changes in functions of these, making, therefore, impossible the precise measurements of cellular functions.

 The volume of liquid to be measured, as described above, refers to a total volume of liquids used when the various measurements described below are made, for example a sum of a volume of blood to be measured, a volume of an anticoagulant solution described below added when necessary, a volume of a blood coagulation enhancer solution, and a volume of liquid to dissolve or suspend stimulators.

 It should be understood, in this case, that it is not necessary that the volume amount of the endotoxin-free water when used to perform the extraction described above be exactly equal to the volume of liquid to be measured, and may be less than the volume of liquid to be measured as long as the extraction can be carried out successfully. Even in such a case, the function and effect of the present invention can be obtained, provided that the endotoxin content in the extracted solution does not exceed 0.5 EU / ml.

 Although there are various methods for determining the content of the above endotoxin, a method mentioned herein to determine the content of endotoxin is a colorimetry, according to the document "Endotoxin testing method" in the vademecum of the 13th revised Japanese Pharmacopoeia . Colorimetry uses, as an indication, a color produced on a synthetic chromophore substrate when it is hydrolyzed, and can be performed, for example, using a commercially available product ENDOSPECIE (manufactured by Seikagaku Kogyo Co.).

 As a method of removing or deactivating an endotoxin, various well-known techniques may be employed that include deactivation by heating, treatment with acid or alkali; ultrafiltration using a membrane filter; and removal using anionic chitosan resins, polymyxin B that can specifically bind endotoxin, or adsorbents that bind antibodies against endotoxin. In addition, the instruments and containers used for endotoxin removal operations are subjected to dry heat treatment at 250 ° C for an hour or more, if made of 30 glass, or immersed in a 0.2 M aqueous solution of sodium hydroxide and washed with endotoxin-free water, if made of plastic, to ensure complete deactivation of endotoxin before use. In addition, endotoxin-free water should be used systematically when water is needed, and an operating atmosphere is preferably an atmosphere such that any secondary endotoxin contamination is limited within a feasible range, as provided in a clean room.

 An interior of the container for measuring cellular functions, according to the invention, is preferably subjected to vacuum. The internal pressure can be reduced to such an extent that atmospheric blood can be suctioned into the interior of the vessel for the measurement of cellular functions after communication thereof with the interior of the vessel for the measurement of cellular functions. The internal pressure can be determined depending on a quantity of blood to be sucked. That is, the greater the amount of target blood to be sucked, the greater the degree of pressure reduction may have to be performed.

 The shape of the first container for the measurement of cellular functions, in accordance with the invention, is not particularly specified, and can be tubular as exemplified by blood collection tubes, test tubes and the like, or similar to a plate as represented by microplates. Preferably, it is suitable for vacuum operation.

 They are illustrative of the type of material for the container for measuring glass and plastic cell functions. Thermoplastic and thermosetting resins can both be used as plastics mentioned above. Examples of thermoplastic resins include polyethylene, polypropylene, polystyrene, polymethylmethacrylate, polyvinyl chloride, polyethylene terephthalate, styrene-acrylonitrile copolymer, styrene-maleic anhydride copolymer, styrene-acrylamine copolymer, ethylene acrylate copolymer, copolymer -propylene, ethylene-acrylic acid copolymer, and ethylene-acrylate copolymer. Examples of thermosetting resins include unsaturated polyester resins, epoxy resins and epoxy acrylate resins.

 In the case where a tubular part is used as the first container for the measurement of cellular functions, according to the invention, a plug is generally used to keep the interior of the container at a reduced pressure. Exemplary types of material for the cap include butyl gums, chlorinated butyl gums, thermoplastic elastomers and the like.

 If the tubular part is used as the first container for the measurement of cellular functions, according to the invention, the amount of blood to be collected varies depending on a volume of the container for the measurement of cellular functions, but can generally be of approximately 0.5-2 ml in case a container having a volume of 4-5 ml is used.

 On the other hand, if a part similar to a microplate is used as a container for the measurement of cellular functions, according to the first invention, the amount of blood to be collected can be approximately 0.05-2 ml.

 Anticoagulant is introduced into the container for the measurement of cellular functions, according to the invention, to prevent blood coagulation. The anticoagulant described above may be present in liquid or solid form in the container. As the above anticoagulants, heparin compounds, citric acid compounds, oxalic acid compounds and the like are mentioned. Sodium heparin is more preferable because it does not inhibit the biological reactions of cells. When blood is collected in the container, a reduced concentration of sodium heparin in the blood possibly causes blood clotting while a higher concentration of it is likely to cause unexpected activation or inactivation of the cells. Accordingly, a preferred amount of sodium heparin housed in the container is 4-50 U / ml. twenty

 It is necessary that the anticoagulant be added to the interior of the first container for the measurement of cellular functions, according to the invention, in particular in the case where the determination is made as for physiologically active substances produced (released or induced) by reacting to cells, such as monocytes, macrophages, lymphocytes, leukocytes or the like, with material capable of inducing the production of physiologically active substances.

 In addition, when necessary, coagulant can be housed in the container for the measurement of cellular functions according to the invention. Such a case may arise when physiologically active substances are determined by blood coagulation. Thrombin and the like can be mentioned as examples of said coagulants.

 An exemplary method of manufacturing the container for the measurement of cellular functions, according to the invention, will be explained below in reference to the manufacture of the tubular container. This is manufactured by adding anticoagulant or coagulant, depending on what is necessary, in a tubular container that has an interior whose pressure can be reduced and in which an amount of endotoxin, when extracted by collecting endotoxin-free water of a volume equal to The volume of liquid to be measured is translated into an insufficient level to induce the production of 5 physiologically active substances from blood cells; bringing the container to a predetermined vacuum state; and placing a cap on the container.

 As the above container, a closed end tubular body is preferred, for example, which is open at one end and closed at the opposite end 10. An opening part is preferably configured to be blocked by a plug body. In addition, the first measuring vessel according to the invention is preferably manufactured in an atmosphere as clean as possible to avoid infection with endotoxins or various bacteria, and is preferably subjected to well-known sterilization treatment after completion of its manufacture, if it's possible.

 In the use of the vessel for the measurement of cellular functions according to the invention, the vessel for the measurement of cellular functions specified above is first placed in communication with a blood vessel to allow blood to be suctioned into the interior. of the container for the measurement of the previous cellular functions. In the communication of the previous blood vessel with the vessel for the measurement of the cellular functions specified above, an injection needle can be used which is used for conventional vacuum blood collection techniques, that is, a needle (generally referred to as a multiple injection needle). ) which has at one end of a needle base a needle part to stick in the blood vessel and on the other side of the needle base another needle part to stick in the cap in the container specified above, and in the one that the needle part for sticking in the blood vessel described above communicates with another needle part for digging into the cap.

 The determination of physiologically active substances in blood is performed by determining the physiologically active substances in blood collected by using the container for the measurement of cellular functions. In this case, the blood collected through the use of the first container for the measurement of cellular functions according to the invention is transferred to another container or subjected to the measurement of physiologically active substances by successive use of the container. In such a case that the determination of the physiologically active substances is carried out by the successive use of the container, the container, after the collection of blood, is generally allowed to stand or centrifuged to separate the hemocytes and plasmas containing physiologically active substances. which are quantitatively determined by reagents for their respective quantification.

 As examples of the above physiologically active substances, TNF-α is mentioned; interleukins such as IL-1β, IL-4, IL-5 and IL-6; interferons such as INFα, INFβ and INFγ; colony stimulating factors; chemotactic factors such as IL-8, RANTES and various cytokines; prostaglandins; PGE2 and PGI2; leukotrienes such as LTB4 and LTC4; various chemical mediators such as nitrogen monoxide, active oxygen, histamines, and platelet activating factor (PAF). Adhesion factors such as soluble ICAM1, soluble cytokine receptors such as soluble IL-2 receptor, matrix metalloproteinases, and intracellular granular enzymes such as macrophage-specific elastase are also mentioned. twenty

 As an exemplary method of determining the physiologically active substances described above, an enzymatic immunoassay using monoclonal or polyclonal antibodies against its target physiologically active substances, peroxidases, enzymes such as alkaline phosphatases, and chromophore substrates of respective enzymes is mentioned. 25

 As exemplary materials capable of inducing the production of physiologically active substances, various microorganisms such as endotoxin, dead BCG bacteria and Corynebacterium are mentioned; synthetic lipid A; pyran copolymer; lectins (such as phytohemagglutinin, concanavalin A, pathogen mitogen); OK432 (Picibanil); PSK 30 (Krestin); lentinan, zymosan; LPS (lipopolysaccharide); calcium ionophore; forbol esters; vehicles fixed to immunoglobulin; formylpeptides such as formyl-methionyl-leucyl-phenylalanine (FMLP); and various cytokines.

 Specific antigens (for example, household powders; mite antigens; various pollen antigens such as 35 Artemis pollen extracts, cedar pollen extracts, rice extracts and the like; fungal antigens; parasitic antigens such as extracts may also be used). of the genus Ascaris and the like; food antigens such as ovalbumin, wheat, soy, lobster, crab, meat and the like; and wasp toxin) that are used for the examination of allergens such as asthma, 5 polynose, allergic rhinitis, atopic dermatitis , gastrointestinal allergy, parasitic allergy and the like.

 Also mentioned are materials that fix the materials illustrated above capable of inducing the production of physiologically active substances in various natural or synthetic high molecular weight materials, 10 by well known fixing techniques (such as covalent bonds, physical adsorption and the like).

 The form of the materials capable of inducing the production of physiologically active substances described above is not particularly limited, and may be in liquid or particulate form, for example. 15 In addition, they can be fixed on vehicles. In the case of the liquid form, the materials described above capable of inducing the production of physiologically active substances are generally diluted for use, such as using, as diluent liquid, a buffer solution such as phosphate buffer, Hanks buffer or the like, a normal culture medium such as 20 MEM, RPMI-1640 or the like, a physiological saline solution (for example, manufactured by Otsuka Seiyaku Co.), or water for injection (for example, manufactured by Otsuka Seiyaku Co.).

 The existing form of the material capable of inducing the production of physiologically active substances described above may be solid or liquid. In the case in which said material capable of inducing the production of physiologically active substances is soluble in water, it may be extended on a surface of the inner wall of the container or added to the container before transforming it into a powder form. For example, in the case where the material capable of inducing the production of physiologically active substances 30 is diluted with water for injection, it is preferred to introduce the material capable of inducing the production of physiologically active substances in the container for subsequent dry solidification of the same. In the case where the material capable of inducing the production of physiologically active substances is insoluble in water, said water-insoluble material may be left preferably to remain immersed in the diluent liquid described above, for example, since it is likely that the possible bubble retention on a surface of the material capable of inducing the production of physiologically active substances causes excessive hemolysis when brought into contact with blood, such as by flip mixing, 5 to result in an adverse influence on a measurement system .

 In the case where the material capable of inducing the production of physiologically active substances is fixed to a vehicle, if the fixing vehicle is a water soluble material, said water soluble material may be extended on an internal wall of the container or add to container 10 before transforming it into powder form. If the fixing vehicle is a water-insoluble material, it can preferably be allowed that said water-insoluble material remains submerged in the diluent liquid described above, for example, since it is likely that the possible retention of bubbles on a surface of the material insoluble in Water causes excessive hemolysis when placed in contact with the blood, such as by flip mixing, to result in an adverse influence on a measurement system.

 It is preferred that the amount of the material capable of inducing the production of physiologically active substances for addition to the container is properly adjusted to an optimum concentration level, depending on the type of material capable of inducing the production of physiologically active substances.

 In the method for measuring cellular functions according to the first invention, in the case where another reactor is prepared separately from the container of the first invention, the shape of the other reactor is not specifically specified, and can be tubular as exemplified by blood collection tubes, test tubes and the like, or similar to a plate as exemplified by microplates and the like. The reactor is preferably adapted so that an amount of endotoxin, when extracted by collecting endotoxin-free water of a volume equal to the volume of liquid 30 to be measured, results in an insufficient level to induce the production of physiologically active substances. from blood cells. In this case, the measurement of the volume of liquid described above that will be subjected to measurement is equivalent to that given above in the explanation of the container for the measurement of cellular functions.

 In the case where the reactor is prepared separately from the vessel for the measurement of cellular functions, as described above, said reactor can be used to which the material capable of inducing the production of physiologically active substances has been added, and which has been brought to a predetermined empty state. For said reactor, its communication with the vessel for the measurement of the cellular functions in which the blood has been collected, such as that provided by the multiple injector, facilitates the transfer of the collected blood to the reactor. In addition, the preload of material capable of inducing the production of physiologically active substances simplifies a reaction process.

 In the above case, it is preferred that the amount of material capable of inducing the production of physiologically active substances for addition to the reactor is properly adjusted to an optimum concentration level, depending on the type of material capable of inducing the production of physiologically active substances. active. An exemplary method of manufacturing this reactor is mentioned below. The material capable of inducing the production of physiologically active substances and, if necessary, the anticoagulant is added to a tubular container that has an interior whose pressure can be reduced. Next, the reactor is brought to a predetermined vacuum state 20 before the plug is placed therein. The reactor for use in the present invention is preferably manufactured in an atmosphere as clean as possible to avoid infection with endotoxin or various bacteria, and is preferably subjected to a well-known sterilizing treatment after completion of its manufacture, if possible. 25

 Like the reactor mentioned above, a closed end tubular body is preferred, for example, which is open at one end and closed at the opposite end. The opening part is preferably configured to be blocked successfully by a plug body. The front closed end tubular body is preferably a body 30 similar to a test tube suitable for the operation of a post-reaction centrifuge, for the measurement of the material capable of inducing the production of physiologically active substances described above, and its Preferred size is 5-30 mm in external diameter and 20-150 mm in height. 35

(Second vessel for the measurement of cellular functions)

 The second container for the measurement of cellular functions according to the invention is characterized in that a material, capable of inducing the production of physiologically active substances in blood when it comes into contact with blood, is housed inside such that it can 5 come into contact with blood. This material capable of inducing the production of physiologically active substances can also be referred to hereinafter as a material that induces physiologically active substances. As the physiologically active substances, cytokines are mentioned. 10

 The inducing material of physiologically active substances is an endotoxin. Endotoxin acts on monocytes and macrophages in blood to promote the activation of these cells and induce the production of cytokines. As the above endotoxin is mentioned the endotoxin constituted by cell wall polysaccharide (LPS) obtained from microorganisms, for example. In addition, materials that fixed endotoxin in various natural or synthetic high molecular weight materials can be used by a fixation technique.

 The amount of endotoxin use is selected so that a concentration of endotoxin in a complete liquid (a sum of blood, an anticoagulant solution, a solution with dissolved endotoxin and so on) when contacted with blood is preferably within 0 , 6 - 100,000 EU / ml, more preferably within 0.8 - 80,000 EU / ml. As the concentration falls below 0.6 EU / ml, the amount of TNFα, IL-1β and IL-6 induced possibly becomes excessively small. As the concentration exceeds 100,000 EU / ml, the amount of TNFα, IL-1β and IL-6 induced possibly becomes excessively small. This is also added to the cost.

 In the second container for the measurement of cellular functions according to the invention, the physiologically active substance inducing material described above is provided in such a way that they can come into contact with the blood within the container. Said state so that it can come into contact with blood is, for example, when the physiologically active substance inducing material is housed in the container.

 The general form of the inducing material of physiologically active substances is a powder form or a liquid form taken when the inducing material is dissolved in a solvent such as water. The state of the inducing material present in the container can be a solid, gel or liquid form. In the case of water-soluble inducing material, it can be dissolved in a suitable solvent to subsequently spread it on a surface of the inner wall of the container or the addition to the container before converting it into a powder form.

 Any of the buffers such as phosphate buffer, Hanks buffer and the like, and normal media such as MEM, RPM-1640 and the like can be used as the solvent described above, provided it is a physiological buffer. In addition, a commercially available injection water (water without LPS, manufactured by Otsuka Seiyaku Co.) as well as physiological saline solution (manufactured by Otsuka Seiyaku Co.) can be used.

 In addition, the amount of inducing material of physiologically active substances incorporated in the second container for use in the invention must be regulated before use so as not to influence the measured values of the above inductively produced physiologically active substances. As is evident from the EXAMPLES described below, as the endotoxin content such as LPS increases, a marked induction of cytokines occurs. Therefore, in order to accurately perform the measurement of cellular functions according to the invention, the content of the inducing material of physiologically active substances must not be greater than the level insufficient to induce cytokines.

 The endotoxin content described above, when endotoxin-free water of a volume equal to a volume of liquid to be subjected to measurement is collected in the container for subsequent extraction with stirring at 37 ° C for one hour, in the same manner as in the first container is preferably made not exceeding 0.5 EU (international endotoxin unit) / ml, as a concentration in the extracted solution.

 Various techniques described in the explanation of the first container 30 can be used as methods to remove or deactivate the endotoxin.

 In addition, since the second container for the measurement of cellular functions, according to the invention, is used to measure functions of cells present in the blood, the anticoagulant may preferably be housed in the previous container to prevent coagulation 35 of the blood.

 The existing form, type and load of the previous anticoagulant are the same as in the case of the first container for the measurement of cellular functions according to the invention. In addition, as regards the types of material of the container for the measurement of cellular functions according to the present invention, those that are the same as for the first container for the measurement of cellular functions according to the invention.

 In addition, in the second container for the measurement of cellular functions according to the invention, an interior thereof is preferably in a vacuum, thus allowing an easy suction of blood into the container for the measurement of cellular functions. according to the second invention. In such a case, the use of a stopper to keep the interior at a reduced pressure is desired, similar to the case of the first container for the measurement of cellular functions according to the invention, and various materials illustrated in the explanation of the First container for measuring cell functions according to the invention can be exemplified as types of plug material.

 The amount of blood collected in the measurement of cellular functions using the second vessel for the measurement of cellular functions according to the invention depends on a volume of the vessel for the measurement of cellular functions, but approximately 0.5 - 2 ml is sufficient when the container for measuring the cellular functions used has a volume of 4-5 ml.

 As an exemplary method of manufacturing the second container for the measurement of cellular functions according to the invention, a method is mentioned in which the physiologically active substance inducing material described above, as well as the anticoagulant, are added to a container which It has an interior whose pressure can be reduced, the container is brought to a predetermined vacuum state, and the cap is placed in the container. 30

 As the container described above, a closed end tubular body is preferred, for example, which is open at one end and closed at the opposite end. The opening part is preferably configured to be blocked by a plug body. More preferred than the anterior closed end tubular body is suitable for operation of a centrifuge performed after the cytokine induction reaction and to determine the amount of induced cytokines, and its preferred size is 5-30 mm in external diameter. and approximately 20 - 150 mm high.

 Furthermore, the container for the measurement of cellular functions according to the invention is preferably manufactured in an atmosphere as clean as possible to avoid infection with endotoxin or various bacteria, and is preferably subjected to a well-known sterilizing treatment after completing its manufacturing, if possible.

 Next, a method of measuring cell functions using the second container for measuring cell functions according to the invention will be explained.

 First, the cell function measuring vessel described above is put in communication with a blood vessel or a blood collection vessel so that a blood sample is sucked into the container for the measurement of functions cell phones. A moderate agitation is then applied to the vessel for the measurement of cellular functions to bring blood cells into contact with the physiologically active substance inducing material described above for a subsequent inductive reaction. As the reaction ceases, the vessel is allowed to stand or centrifuged to separate hemocytes and plasmas, and then the cytokines in the plasmas are quantitatively determined by reagents capable of quantifying respective cytokines.

 The technique described above for communicating the first vessel for the measurement of cellular functions with the vessel for blood collection can be used to communicate the vessel for the collection of blood described above with the vessel for the measurement of blood Cellular functions described above.

 If the temperature at which the blood is reacted with the previous cytokine inducing material is lowered, the metabolic activity of the cells 30 is possibly lowered to result in a reduced excess amount of induced cytokines, and if it rises, possibly cell damage occurs to result in an excessively reduced amount of induced cytokines. Therefore, it is preferably controlled at 26-45 ° C, more preferably at 30-42 ° C. 35

 If the period of time during which the blood is reacted with the previous cytokine inducing material is shortened, the amount of induced cytokines becomes possibly excessively small, and if this is prolonged excessively, the production of the measurement results is delays In addition, the amount of induced cytokines shows a tendency to be gradually reduced from a maximum that takes place to approximately 4 hours. The preferred period of time is therefore 1-6 hours, more preferably 2-4 hours.

 In the method of measuring the cellular functions that the second container uses for the measurement of the cellular functions according to the invention, it is most preferred that a complete liquid collected in the container for the measurement of the cellular functions is cultured at 30-40 ° C for 2-6 hours to induce cytokines.

 The enzyme immunoassay described in the explanation of the container for the measurement of cellular functions according to the invention can be used to quantitatively determine the induced cytokines.

 An embodiment of a technique for measuring cellular functions using the second vessel for the measurement of cellular functions according to the invention will be explained in detail below. First, the cytokine inducing material described above is reacted with blood in the vessel for the measurement of the cellular functions described above to induce cytokines. Once the induction is complete, the cell function measuring vessel is centrifuged at 1200 G to separate the hemocyte components and the plasma components. Then, the separated plasmas are added using a pipette to a well of a 25 microplate in which monoclonal anti-cytokine antibodies have been fixed for a subsequent reaction at 37 ° C for 2 hours. Then, the plasma solution after the reaction was removed by means of suction removal or the like, and in addition, the well is washed with a neutral pH wash buffer containing a non-ionic surfactant, such as Tween 20, to additionally remove components that did not react. Polyclonal anti-cytokine antibodies bound to peroxidases of horseradish peroxidases are then added to the well for a reaction at 37 ° C for 1 hour. The well is then washed with the previous wash buffer to remove the unreacted horseradish peroxidases, and then a substrate solution containing hydrogen peroxide and tetramethylbenzidine is added for the reaction for 5-10 minutes. A 1 M solution of sulfuric acid is added to interrupt the reaction before determining a color produced in the substrate due to an enzymatic reaction from absorbance at 450 nm. The determined value is evaluated against a calibration curve prepared using cytokines of known concentration to determine the level of induced cytokines.

(Kit for measuring cell functions)

 Each of the first and second containers for the measurement of cellular functions according to the invention is combined with a reagent 10 capable of quantifying physiologically active substances, that is to say an enzyme immunoassay reagent, to provide a usable kit for the measurement of cellular functions That is, a kit is provided for the measurement of the cellular functions that the first container has for the measurement of the cellular functions and the reagent capable of quantitatively determining the 15 physiologically active substances induced and also has the second container for the measurement of the cellular functions and the reagent capable of quantitatively determining the induced physiologically active substances.

(Preferred anticoagulants)

 In the present invention, the amount of material capable of inducing the production of physiologically active substances contained in the anticoagulant described above is ideally controlled at an insufficient level to produce physiologically active substances from blood cells when mixed with blood. That is, the reduction of the amount of the physiologically active substances originally contained in the anticoagulant effectively limits the production of unnecessary stimulation given to the blood collected before the test. The production of physiologically active substances in the collected blood due to the action of the anticoagulant is regulated in this way, so that the determination of various physiologically active substances, the measurement of cellular functions, and the determination of cell surface antigens Blood can be performed more accurately. In addition, a blood sample can be stored for a prolonged period of time from collection to testing.

 Again in such a case, the physiologically active substance inducing materials may be those described above, and one preferred in an endotoxin. The increased endotoxin content causes the production of the aforementioned cytokines, such as TNFα, IL-1β, IL-6 and others, to interfere with an accurate measurement. Therefore, the endotoxin content in the anticoagulant is ideally regulated at an insufficient level to produce cytokines, as physiologically active substances, in the collected blood.

 As will be apparent from the Examples described below in this document, the production of cytokines, such as TNαF, IL-1β, IL-6 and others, is possibly induced if the endotoxin content in the anticoagulant exceeds 0, 5 EU / ml in reactive blood. Therefore, it is desired to regulate the endotoxin content in anticoagulant so that the endotoxin content in a reactive liquid does not exceed 0.5 EU / ml.

 The amount of the anticoagulant described above depends on the amount of blood to be collected, but is generally 0.5-5 mg / ml in blood, if ethylenediamine sodium tetraacetate is used, 3-5% by weight in blood is used , if sodium citrate is used, and 4-50 U / ml in blood, if sodium heparin is used.

 Accordingly, it is preferred in the present invention that the endotoxin content in anticoagulant can be suitably selected depending on the amounts respectively of anticoagulant and blood collected, so that the endotoxin content in blood collected results in a level that does not exceed 0.5 EU / ml.

 For example, when 1 ml of blood is collected for examination, sodium heparin, if selected for use, is generally added in an amount of 4-50 U / ml, and therefore the preferred endotoxin content thereof is not it exceeds 00,125 EU / unit of heparin, more preferably it does not exceed 0,01 EU / unit of heparin.

 The various techniques described in the explanation of the invention for removing or deactivating endotoxin, for example, can be used to make anticoagulants containing a reduced amount of endotoxin.

 However, the inactivation of endotoxin with heat, acid or alkali treatment sometimes accompanies the deactivation of a certain anticoagulant itself. Therefore, ultrafiltration using a membrane or removal using an adsorbent is preferred. 35

(Measurement of cellular functions)

 The expression "measurement of cellular functions" as used in the present invention is intended to include a method of directly measuring the functions of a blood cell that can be classified into an erythrocyte, a platelet and a leukocyte, a method of evaluating the 5 cellular functions by determining physiologically active substances, and measuring blood cell surface antigens.

 The measurement of blood cell functions that is classified in the erythrocyte, platelet and leukocyte mentioned above include hemagglutination, platelet agglutination, leukocyte migration, leukocyte migration inhibition assay, leukocyte reduction in tetrazolium nitro blue, phagocytic leukocyte activity, lymphocyte transformation, cytotoxic lymphocyte assay, antibody-dependent cell-mediated cytotoxic activity assay, cytokine production capacity, histamine release assay and the like, for example. fifteen

 In addition, the measurement of cell surface antigens refers to the measurement of cell surface antigens by a rosette formation assay or flow cytometry, and includes the measurement of Fc receptors and various CD antigens, for example.

(Kit for measuring cell functions according to the invention) 20

 The kit for measuring cell functions according to the invention has the first container for measuring cell functions described above, the second container for measuring cell functions and the reagent as defined in claim 1.

 In order to measure the cellular functions using the kit for the measurement of the cellular functions according to the invention, the second container for the measurement of the cellular functions according to the invention is put in communication with a blood collection vessel, a blood sample is introduced into the second vessel for the measurement of cellular functions according to the invention, and then the second vessel 30 for the measurement of cellular functions according to the invention is stirred to react to the blood cells with the inducing material of physiologically active substances.

 In addition, to obtain a control value, the blood collection vessel is placed in communication with the first container for the measurement of the cellular functions according to the invention to introduce the blood sample into the first container for the measurement of cellular functions according to the invention.

 Then, the first and second containers for measuring cell functions, in which blood has been introduced in a manner as described above, are allowed to stand or centrifuged to separate the hemocytes and plasmas, and the Physiologically active substances in plasmas in the respective containers for measurement of cellular functions are determined quantitatively separately by using the first and second enzymatic immunoassay reagents having 10 different respective measuring sensitivities.

 Communication of the blood collection vessel with the respective containers for the measurement of cellular functions according to the invention can be achieved using the technique described above.

 As the reaction temperature of blood and endotoxin 15 in the second vessel for the measurement of cellular functions according to the invention decreases, a reduced metabolic activity of the cells occurs, as well as a reduced cytokine induction. As it increases, cell injury and reduced induction of cytokines occur. Accordingly, this is preferably 26-45 ° C, more preferably 30-42 ° C.

 In view of the effective production of cytokines and the prevention of excessive hemolysis, the reaction period of blood and endotoxin is preferably 1-12 hours, more preferably 2-6 hours.

 In the kit for the measurement of cellular functions according to the present invention, the physiologically active substances in the blood collected in the container for the measurement of cellular functions according to the invention, that is, the physiologically active substances produced in the blood due to its reaction with the inducing material of physiologically active substances is quantitatively determined by means of the second enzyme immunoassay reagent. Physiologically active substances produced in blood, due not to their reaction with the physiologically active substance inducing material, are quantitatively determined by the first enzyme immunoassay reagent. The resulting differential allows the determination of the exact production of 35 physiologically active substances induced by the inducing material of physiologically active substances.

 Generally, the amount of cytokines in blood (the amount of cytokines in blood collected in the first container for the measurement of cellular functions) before your reaction with endotoxin is several pg / ml - 5 several hundred pg / ml, and The amount of cytokines in blood after your reaction with endotoxin varies between several hundred pg / ml and several thousand pg / ml or higher.

 However, there is no reagent such that it has sufficient measurement sensitivity to quantify cytokines over a wide range of several 10 pg / ml - several thousand pg / ml. Therefore, for the determination of cytokines in an amount of 1,000 pg / ml or greater, plasmas were diluted with a suitable dilution solution. However, the plasma dilution operation is complicated, requires the dilution solution and a dilution vessel separately, and therefore leads to a markedly prolonged measurement period. In addition, the measured value is obtained by multiplying the degrees of dilution, which raises the problem of reducing the accuracy of the measured value.

 On the contrary, according to the invention, the amount of cytokines in blood (i.e., the amount of cytokines in blood collected in the first container for the measurement of cellular functions) before their reaction with endotoxin can be determined by the first high sensitivity enzyme immunoassay reagent, such as a measurement sensitivity of 10-1000 pg / ml, while the amount of blood cytokines (i.e., the amount of blood cytokines collected in the second container 25 for measurement of cellular functions) after its reaction with endotoxin can be determined by the second low sensitivity enzyme immunoassay reagent, such as a measuring sensitivity of 500-10,000 pg / ml. In this way, the complicated dilution operation described above can be eliminated. 30

 In addition, if the cytokine described above is constituted by TNFα or IL-β, the preferred sensitivity of the first enzyme immunoassay reagent is 10-500 pg / ml and that of the second enzyme immunoassay reagent is 500-10,000 pg / ml, if it is IL-6, the preferred sensitivity of the first enzyme immunoassay reagent is 10-1000 pg / ml and that of the second enzyme immunoassay reagent is 1,000-20,000 pg / ml.

 Each of the first and second immunoassay reagents described above is composed of a monoclonal or polyclonal antibody against its target cytokines, enzymes such as peroxidases or alkaline phosphatases, and chromophores substrates in respective enzymes. The "sandwich" enzyme immunoassay, in which the monoclonal antibody against its target cytokines is preset on a solid surface such as that of a microplate, is preferred, since it does not require fixation before measurement and has excellent reproducibility.

 A technique of fixing the monoclonal antibodies on solid surfaces 10 can be arbitrarily selected from the known physical adsorption technique or chemical bond, but physical adsorption is preferred by its simple operation.

 The preparation of the first and second enzymatic immunoassay reagents having different sensitivities can be performed by selectively adjusting concentrations of monoclonal or polyclonal antibodies respectively against target cytokines, enzymes such as peroxidases or alkaline phosphatases, and chromophores substrates in respective enzymes.

 For example, in the case where the "sandwich" enzyme immunoassay is used, the amount of the aforementioned monoclonal antibody 20 against its target cytokines for fixation on the microplate can be adjusted, so that the first and second reagents of Enzyme immunoassay that have different sensitivities. That is, the amount of target cytokines that can bind to monoclonal antibodies changes depending on the amount of monoclonal antibody fixed on the surface of the microplate, which allows the preparation of reagents that have different measurement sensitivities.

 Said different measurement sensitivities can also be achieved by preparing different concentrations of enzyme-labeled monoclonal antibody or enzyme-labeled polyclonal antibody against its target cytokines, which differ from the target cytokines of the fixed monoclonal antibody, as a reagent to detect cytokines bound to the fixed monoclonal antibody .

 There is also a method in which a specific binding mode such as avidin-biotin is incorporated into the enzyme immunoassay system 35 described above, for example a method in which biotin-labeled antibody or / and avidin-labeled enzyme is used as an alternative to the enzyme-labeled antibody against its target cytokine that differs from the target cytokines of the fixed monoclonal antibody.

 An exemplary preparation of the microplates with fixed monoclonal antibody 5 will be explained below, which can be used for the present first and second enzyme immunoassay reagents having different measurement sensitivities.

 First, the specific monoclonal antibody against its target cytokines is dissolved in a buffer solution, such as phosphate buffer, to prepare two types of dilute solutions of different concentration levels (each prepared concentration level is properly selected depending on the magnitude of the binding constant of the monoclonal antibody used and its target cytokines).

 Each of the two types of solutions with dissolved monoclonal antibody is added in a given amount to a microplate for incubation at 2-8 ° C for one day and one night. After washing with a neutral pH wash buffer containing a non-ionic surfactant such as Tween 20, a given amount of phosphate buffer solution with 1-4% bovine serum albumin in dissolved weight is added to the microplate for incubation at 20 37 ° C for 2 hours. After removal of the liquids from the microplate, it is dried at room temperature.

 An embodiment of measuring cell functions using the kit for measuring cell functions according to the invention will be explained in detail below. 25

 First, blood is introduced into each of the first and second vessels for measuring cell functions for incubation at 37 ° C for 4 hours. Each vessel is centrifuged at 1,600 G to allow hemocytes and plasmas to separate. Next, the separated plasmas are added to the wells of respective microplates at which the monoclonal antibody against their target cytokines has been fixed at two different concentration levels, which have been blocked at sites not adsorbed by bovine serum albumins, and that have dried, for reaction at 37 ° C for 2 hours. Then, the plasma solution after the reaction is discarded by means of suction removal and the like, followed by washing the wells with a neutral pH wash buffer containing a non-ionic surfactant such as Tween 20 to further remove the components that did not react. Next, the polyclonal antibody bound to horseradish peroxidase against the cytokines specified above is added to the wells for reaction at 5 37 ° C for 1 hour. To remove the unreacted portion of polyclonal antibody bound to horseradish peroxidase, the wells are washed with the wash buffer mentioned above, and then a substrate solution containing hydrogen peroxide and tetramethylbenzidine is added to the wells for reaction during 5 - 10 minutes. A solution of 2M sulfuric acid is then added, the reaction is interrupted, and a color produced in the substrate as a result of the enzymatic reaction is measured from absorbance at 450 nm. The comparison of the measured value with a calibration curve prepared against the cytokines specified above of a known concentration quantitatively determines the blood cytokines 15 as treated in each of the first and second vessels for the measurement of cellular functions.

(Method for measuring cellular functions)

 The method for measuring cell functions is characterized by introducing blood into the first or second container for measuring cell functions and measuring cell functions. In this case, the container for the measurement of cellular functions preferably houses, in advance, the anticoagulant specified above containing endotoxin in such a limited concentration as not to induce blood cells to produce physiologically active substances 25 when mixed with the blood.

 An embodiment of the method for measuring cellular functions is explained below.

 The anticoagulant, for example sodium heparin is housed in an injector that has a needle for blood collection in a typical amount 30 of 10 U / ml for blood to be collected, before the blood is collected from a person examined inside of the injector.

 As an alternative, the anticoagulant can be housed in a tube for vacuum blood collection in the same amount as before before blood collection. This blood is then centrifuged at 35,600 G, and the amount of TNFα, as an illustration of the physiologically active substance produced from blood cells, in plasma is determined using the enzyme immunoassay.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing measurement results obtained in Examples 1-4 and Comparative Examples 2-4, in which the abscissa axis indicates the concentration of LPS and the ordinate indicates the amount of induced TNFα.

Figure 2 is a graph showing measurement results obtained in Examples 1-4 and Comparative Examples 2-4, in which the axis 10 of abscissa indicates the concentration of LPS and that of ordinates indicates the amount of IL-1β induced.

Figure 3 is a graph showing measurement results obtained in Examples 1-4 and Comparative Examples 2-4, in which the abscissa axis indicates the concentration of LPS and the ordinate axis indicates the amount of IL-6 induced.

Figure 4 is a graph showing measurement results obtained in Examples 5-10, in which the abscissa axis indicates the concentration of LPS and the ordinate indicates the amount of TNFα or IL-1β induced. twenty

Figure 5 is a graph showing measurement results obtained in Examples 11-16, in which the abscissa axis indicates the reaction temperature and the ordinate axis indicates the amount of TNFα or IL-1β induced.

Figure 6 is a graph showing measurement results obtained in Examples 17-21, in which the abscissa axis indicates the reaction time and the ordinate axis indicates the amount of TNFα or IL-1β induced.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

 The present invention will be explained in detail below by citing Examples of the present invention and Comparative Examples. Reference Examples are not within the scope of claim 1.

Examples 1-4 (Reference), Comparative Examples 1-4

(1) Method to manufacture a container for the measurement of cellular functions:

 A 4 ml (12.6 x 75 mm diameter) blood collection tube made of polyethylene terephthalate was washed well 10 times with 4 ml of endotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) containing sodium heparin (manufactured by Novo.Nordisk A / S Co., product name: Novo.Heparin # 1000) at the concentration of 200 U / ml was added to the tube for blood collection.

 The endotoxin obtained from E. coli UKT-B fat (standard product lot 8920 of the Japanese Pharmacopoeia) was dissolved in a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) for subsequent stage dilution. Once each dilution stage is completed, 0.05 ml of the resulting saline solution with dissolved endotoxin are added to each of the tubes for blood collection with incorporated heparin, so that the tubes for blood collection are prepared containing respectively endotoxin in concentrations of 0 EU / ml (Comparative Example 1), 1 EU / ml (Comparative Example 2), 2 EU / ml (Comparative Example 3), 5 EU / ml (Comparative Example 4), EU 10 / ml (Example 1), 20 EU / ml (Example 2), 50 EU / ml (Example 3) and 100 EU / ml (Example) for a complete solution that adds the physiological saline solution with dissolved heparin and the physiological saline solution with dissolved endotoxin.

 Next, a cap made of butyl rubber, which was configured to fit into the tube and was previously washed with an endotoxin-free water (manufactured by Otsuka Seiyaku Co.), was placed superimposed on an opening of each tube for collecting blood so as not to tightly close the opening. Next, each blood collection tube was placed inside a vacuum vessel. When the pressure inside the vessel was gradually reduced to a pressure of 570 mm Hg, the opening of each blood collection tube was tightly closed by the cap. The blood collection tubes prepared in this way were used as the containers for the measurement of cellular functions in Examples 1-4 and Comparative Examples 1-4, 30 respectively.

(2) Determination of endotoxin content in blood collection tubes (Containers):

 Endotoxin-free water (manufactured by Otsuka Seiyaku Co.) was introduced into an injector with a needle. The needle of the injector was stuck in the cap made of butyl rubber, placed on each of the four tubes for collecting blood under vacuum that were obtained in the previous one (1) containing heparin alone, to inject 0 , 9 ml of the endotoxin-free water (manufactured by Otsuka Seiyaku Co.) in each of the blood collection tubes, followed by stirring at 37 ° C for 1 hour to extract the endotoxin. Next, the endotoxin content in the extracted liquid was determined by a chromophore substrate technique synthesized using an endotoxin determination kit, ENDOSPECIE ES6 (product name) manufactured by Seikagaku Kogyo Co.

 The results indicated that the endotoxin content in the extracted liquid was not greater than 0.05 EU / ml for each of the four blood collection tubes tested.

(3) Methods to determine the induction activities of TNFα, IL-1β and IL-6

 Using an injector with a needle, heparinized blood was collected from a usually healthy volunteer. The needle of the injector was stuck in the cap made of butyl rubber, placed on each of the tubes for the collection of blood subjected to the vacuum that were obtained in the previous one (1) containing endotoxin at various concentrations, to inject 0 , 9 ml of the blood sample collected in each of the blood collection tubes. Next, each of the blood collection tubes was mounted on a stirring platform for flip mixing in a preheated thermostatic chamber at a temperature of 37 ° C for subsequent mixing by flipping for 4 hours. Once the intimate mixing was completed, each blood collection tube was centrifuged at 1,600 G at 4 ° C for 10 minutes to collect a supernatant plasma. In the collected plasma, the cytokine content (pg / ml) was determined, that is, the respective content of TNFα, IL-1β and IL-6, using an enzyme immunoassay kit using respective monoclonal antibodies against them.

 The PREDICTA Human TNF-α ELISA KIT kits (detection limit: 35 pg / ml), PREDICTA Human IL-1β ELISA KIT (detection limit: 30 15 pg / ml) and PREDICTA Human IL-6 ELISA KIT (limit were used Detection: 35 pg / ml) (all manufactured by Genzyme Inc.) to determine the weight productions of TNFα, IL-1β and IL-6, respectively. The determination was made using n = 3 for each. The results are shown in Figures 1-3.

 In all figures, the EU / ml unit used for the concentration of 35 LPS on the abscissa axis indicates the concentration of endotoxin in a complete solution when in contact with blood, and the amount induced (pg / ml) in the ordinate axis indicates an average value of the values obtained from the two blood collection tubes for each concentration of cytokines in plasma. As is evident from the results, endotoxin at a concentration of 0.6 EU / ml or higher clearly induces the production of TNFα, IL-1β and IL-6. The results of the case in which the concentration of endotoxin was 0 EU / ml (Comparative Example 1) is not shown in Figures 1-3, since, in this case, the concentration of each cytokine produced was below the limit of detection in the determination of it.

EXAMPLES 5 - 10 (Reference)

(1) Method to manufacture a container for the measurement of cellular functions:

 A 4 ml (12.6 x 75 mm diameter) blood collection tube made of polyethylene terephthalate was washed well 10 times with 4 ml of endotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) containing sodium heparin (manufactured by Novo.Nordisk A / S Co., product name: Novo.Heparin # 1000) at a concentration of 200 U / ml were added to the tube. twenty

 The endotoxin obtained from E. coli strain 055: B5 (manufactured by LBL corp.) Was dissolved in a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) for subsequent stage dilution. Once each dilution stage is completed, 0.05 ml of the resulting dissolved endotoxin saline solution is added to each of the tubes for blood collection with incorporated heparin, so that the tubes for blood collection are prepared containing respectively endotoxin in concentrations of 8 EU / ml (Example 5), 80 EU / ml (Example 6), 800 EU / ml (Example 7), 8,000 EU / ml (Example 8), 80,000 EU / ml (Example 9 ) and 800,000 EU / ml (Example 10) for a complete solution that adds the physiological saline with dissolved heparin and the physiological saline with dissolved endotoxin.

 Next, a cap made of butyl rubber, which was configured to fit in the tube and had been thoroughly washed with an endotoxin-free water (manufactured by Otsuka Seiyaku Co.), was placed superimposed on an opening of each tube for collecting blood so as not to tightly close the opening. Next, each blood collection tube was placed inside a vacuum vessel. When the pressure inside the vessel was gradually reduced to a pressure of 570 mm Hg, the opening of each blood collection tube was sealed 5 by the cap. The blood collection tubes prepared in this way were used as the containers for the measurement of cellular functions in Examples 5-10, respectively.

(2) Determination of endotoxin content in blood collection tubes (Containers): 10

 Endotoxin-free water (manufactured by Otsuka Seiyaku Co.) was introduced into an injector with a needle. The needle of the injector was stuck in the cap made of butyl rubber, placed on each of the four tubes for collecting blood under vacuum that were obtained in the previous one (1) containing heparin alone, to inject 0, 9 ml of the free water of endotoxin (manufactured by Otsuka Seiyaku Co.) in each of the blood collection tubes, followed by stirring at 37 ° C for 1 hour to extract the endotoxin. Next, the endotoxin content in the extracted liquid was determined by a chromophore substrate technique synthesized using an endotoxin determination kit, ENDOSPECIE ES6 (name of the product) manufactured by Seikagaku Kogyo Co.

 The results indicated that the endotoxin content in the extracted liquid was not greater than 0.05 EU / ml for each of the four blood collection tubes tested.

(3) Methods to determine the induction activities of TNFα and IL-1β: 25

 Using an injector with a needle, heparinized blood was collected from a usually healthy volunteer. The needle of the injector was nailed in the cap made of butyl rubber, placed on each of the tubes for the collection of blood under vacuum that were obtained in the previous one (1) containing endotoxin at various concentrations, to inject 0, 9 ml of the 30 blood sample collected in each of the blood collection tubes. Next, each of the blood collection tubes was mounted on a stirring platform for flip mixing in a preheated thermostatic chamber at a temperature of 37 ° C for subsequent mixing by flipping for 4 hours. After the intimate mixing was completed, each tube for blood collection was centrifuged at 1,600 G at 4 ° C for 10 minutes to collect a supernatant plasma. In the collected plasma, the content (pg / ml) of TNFα and IL-1β was determined in the same manner as that used in Example 1.

 The results are shown in Figure 4. In Figure 4, the EU / ml 5 unit used for the concentration of LPS on the abscissa axis indicates the concentration of endotoxin in blood (in a complete solution) when in contact with the blood, and the amount induced (pg / ml) in the ordinate axis indicates an average value of the values obtained from the two tubes for blood collection for each of the concentrations of TNFα and 10 IL-1β in plasma. As is evident from the results, the concentration of endotoxin in the range of 0.8-80,000 EU / ml induces productions of TNFα and IL-1β. In addition, when the concentration of endotoxin is in the range of 8-800 EU / ml, the amounts of TNFα and IL-1β induced are indicated as both increase slowly. When the concentration of endotoxin reaches 8,000 EU / ml, the amounts of TNFα and IL-1β induced both indicated an additional increase. However, when the endotoxin concentration reaches 80,000 EU / ml, the amounts of TNFα and IL-1β induced each become smaller than when the endotoxin concentration is EU 8,000 / ml. twenty

 The use of endotoxin, as an illustration of the endotoxin inducing material, in the range of 10-200 µg / ml (approximately 80,000 - 200,000 EU / ml converted to the endotoxin concentration) has already been described in the Open Patent Bulletin a Public inspection No. Hei 1-503331. However, the present invention allows the use of endotoxin at 25 lower concentrations, so that the induction of cytokines by plural mechanisms does not occur, which is believed to be due to a higher concentration of endotoxin. Therefore, in accordance with the present invention, it becomes possible to induce cytokines that accurately reflect the morbidities of the patient. 30

EXAMPLES 11 - 16 (Reference)

(1) Method to manufacture a container for the measurement of cellular functions:

 Each of the 4 ml (12.6 x 75 mm diameter) blood collection tubes made of polyethylene terephthalate was washed well 10 times with 4 ml of endotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) containing sodium heparin (manufactured by Novo.Nordisk A / S Co., product name: Novo.Heparin # 1000) at a concentration of 200 U / ml were added to each tube. 5

 The endotoxin obtained from E. coli strain 055: B5 (manufactured by LBL corp.) Was dissolved in a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) at a concentration of 1,600 EU / ml. 0.05 ml of the resulting dissolved endotoxin saline solution was added to each of the tubes for blood collection with incorporated heparin prepared above.

 Next, a cap made of butyl rubber, which was configured to fit the tube and had been thoroughly washed with an endotoxin-free water (manufactured by Otsuka Seiyaku Co.), was placed superimposed on an opening of each tube to collecting blood so as not to tightly close the opening. Next, each blood collection tube was placed inside a vacuum vessel. When the pressure inside the vessel was gradually reduced to a pressure of 570 mm Hg, the opening of each blood collection tube was tightly closed by the cap. The blood collection tubes prepared in this way were used as the containers for the measurement of cellular functions in Examples 11-16, respectively.

(2) Determination of endotoxin content in tubes for blood collection (Containers):

 Endotoxin-free water (manufactured by Otsuka Seiyaku Co.) was introduced into an injector with a needle. The needle of the injector was stuck in the cap made of butyl rubber, placed on each of the four tubes for collecting blood under vacuum that were obtained in the previous one (1) containing heparin alone, to inject 0, 9 ml of the endotoxin-free water (manufactured by Otsuka Seiyaku Co.) in each of the tubes for blood collection, followed by stirring at 37 ° C for 1 hour to extract the endotoxin. Next, the endotoxin content in the extracted liquid was determined by a chromophore substrate technique synthesized using an endotoxin determination kit, ENDOSPECIE ES6 (product name) manufactured by Seikagaku Kogyo Co. 35

 The results indicated that the endotoxin content in the extracted liquid was not greater than 0.05 EU / ml for each of the four blood collection tubes tested.

(3) Methods to determine the induction activities of TNFα and IL-1β:

 Using an injector with a needle, heparinized blood was collected from a usually healthy volunteer. The needle of the injector was stuck in the cap made of butyl rubber, placed on each of the tubes for the collection of blood subjected to the vacuum obtained in the previous one (1), to inject 0.9 ml of the sample of blood collected in each of the tubes for blood collection. Next, two of the tubes for the collection of blood in which the blood sample had been collected were mounted on each of the stirring platforms for preheated flipping mixing respectively at a temperature of 25 ° C (Example 11), 30 ° C ( Example 12), 33 ° C (Example 13), 37 ° C (Example 14), 40 ° C (Example 15) and 45 ° C (Example 16) in a thermostatic chamber for subsequent mixing by turning 15 for 4 hours. Once the intimate mixing was completed, each blood collection tube was centrifuged at 1,600 G at 4 ° C for 10 minutes to collect a supernatant plasma. In the collected plasma, the content (pg / ml) of TNFα and IL-1β was determined in the same manner as that used in Example 1. 20

 The results are shown in Figure 5. In Figure 5, the reaction temperature on the abscissa axis indicates the temperature at which the thermostatic chamber was adjusted, and the induced quantity (pg / ml) on the ordinate axis indicates an average value of the values obtained from the two blood collection tubes for each of the concentrations of TNFα and IL-1β 25 beta in plasma.

EXAMPLES 17 - 21 (Reference)

(1) Method to manufacture a container for the measurement of cellular functions:

 Each of the 4 ml (12.6 x 75 mm 30 diameter) blood collection tubes made of polyethylene terephthalate was washed well 10 times with 4 ml of endotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) containing sodium heparin (manufactured by Novo.Nordisk A / S Co., product name: Novo.Heparin # 1000) at a concentration of 200 U / ml 35 were added to each tube.

 The endotoxin obtained from E. coli strain 055: B5 (manufactured by LBL corp.) Was dissolved in a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) at a concentration of 1,600 EU / ml. 0.05 ml of the resulting dissolved endotoxin saline solution were added to each of 5 tubes for blood collection with incorporated heparin prepared above.

 Next, a cap made of butyl rubber, which was configured to fit the tube and had been thoroughly washed with an endotoxin-free water (manufactured by Otsuka Seiyaku Co.), was placed superimposed on an opening of each tube for collecting blood so as not to tightly close the opening. Next, each blood collection tube was placed inside a vacuum vessel. When the pressure inside the vessel was gradually reduced to a pressure of 570 mm Hg, the opening of each blood collection tube was tightly closed by means of the cap. The blood collection tubes prepared in this way were used as the vessels for measuring cell functions in Examples 17-21, respectively.

(2) Determination of endotoxin content in blood collection tubes (containers): 20

 Endotoxin-free water (manufactured by Otsuka Seiyaku Co.) was introduced into an injector with a needle. The needle of the injector was stuck in the cap made of butyl rubber, placed on each of the four tubes for collecting blood under vacuum that were obtained in the previous one (1) containing heparin alone, to inject 0, 9 ml of the free water of endotoxin (manufactured by Otsuka Seiyaku Co.) in each of the blood collection tubes, followed by stirring at 37 ° C for 1 hour to extract the endotoxin. Next, the endotoxin content in the extracted liquid was determined by a chromophore substrate technique synthesized using an endotoxin determination kit, ENDOSPECIE ES6 (name of the product) manufactured by Seikagaku Kogyo Co.

 The results indicated that the endotoxin content in the extracted liquid was not greater than 0.05 EU / ml for each of the four blood collection tubes tested.

(3) Methods to determine the induction activities of TNFα and IL-1β: 35

 Using an injector with a needle, heparinized blood was collected from a usually healthy volunteer. The needle of the injector was stuck in the cap made of butyl rubber, placed on each of the 12 tubes for the collection of blood under vacuum that were obtained in the previous one (1), to inject 0.9 ml of the sample of blood collected in each of the tubes for the collection of blood. Next, the blood collection tubes in which the blood sample had been collected were mounted on a stirring platform for preheated flip mixing at a temperature of 37 ° C in a thermostatic chamber for subsequent mixing by flipping for 30 minutes ( Example 17), 2 hours (Example 18), 4 hours (Example 19), 6 hours 10 (Example 20), and 24 hours (Example 21), respectively. In the above Examples, two tubes for blood collection for each Example were subjected to flip mixing during the respective time period specified above. Once the intimate mixing was completed, each blood collection tube was centrifuged at 3,000 rpm at 4 ° C for 10 minutes to collect a supernatant plasma. The content (pg / ml) of TNFα and IL-1β was determined in the collected plasma in the same manner as in Example 1.

 The results are shown in Figure 6. In Figure 6, the reaction time on the abscissa axis indicates the period of time during which the mixing by flip described above was performed, and the induced amount 20 (pg / ml) in the ordinate axis it indicates an average value of the values obtained from the two tubes for the collection of blood for each of the concentrations of TNFα and IL-1β in plasma.

EXAMPLE 22 (Reference)

(1) Method for manufacturing a container for the measurement of cellular functions:

 Each of the 4 ml (12.6 x 75 mm diameter) blood collection tubes made of polyethylene terephthalate was washed well 10 times with 4 ml of endotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.) 30 containing sodium heparin (manufactured by Novo.Nordisk A / S Co., product name: Novo.Heparin # 1000) at a concentration of 200 U / ml were added to each tube.

 Next, sterilized phytohemagglutinin-P (PHA-P) (manufactured by Sigma Chemical Co.) was dissolved in a physiological saline solution for injection (manufactured by Otsuka Seiyaku Co.). 0.05 ml of the resulting physiological saline solution was added to each of the tubes for blood collection with incorporated heparin prepared above, so that each tube for blood collection contained PHA-P in concentrations of 100 µg / ml for a complete solution that adds the physiological saline solution with dissolved sodium heparin and the physiological saline solution with dissolved PHA-P.

 Next, a cap made of butyl rubber, which was configured to fit the tube and had been thoroughly washed with an endotoxin-free water (manufactured by Otsuka Seiyaku Co.), was placed superimposed on an opening of each tube to collecting blood to not tightly close the opening. Next, each blood collection tube was placed inside a vacuum vessel. When the pressure inside the vessel was gradually reduced to a pressure of 570 mm Hg, the opening of each blood collection tube was tightly closed by the cap. Vacuum-drawn blood collection tubes 15 prepared in this way were used respectively for the vessel for the measurement of cellular functions.

(2) Determination of endotoxin content in blood collection tubes (Containers):

 Endotoxin-free water (manufactured by Otsuka Seiyaku Co.) was introduced into an injector with a needle. The needle of the injector was stuck in the cap made of butyl rubber, placed on each of the four tubes for the collection of blood subjected to the vacuum that were obtained in the previous one (1) containing heparin and PHA-P, to inject 0.9 ml of the endotoxin-free water (manufactured by Otsuka Seiyaku Co.) in each of the tubes for blood collection, followed by stirring at 37 ° C for 1 hour to extract the endotoxin. Next, the endotoxin content in the extracted liquid was determined in the same manner as in Example 1. The results indicated that the endotoxin content in the extracted liquid was not greater than 0.05 EU / ml for each of the four tubes for blood collection 30 tested.

(3) Methods to determine the induction activities of TNFα, IL-1β and IL-6:

 The induction activities of TNFα, IL-1β and IL-6 were determined in the same manner as in (3) of Example 1, except that the 35 vacuum-drawn blood collection tubes obtained in the above were used (1 ) containing heparin and PHA-P, instead of using the vacuum-collected blood collection tubes that were obtained in (3) of Example 1 containing endotoxin at various concentrations.

COMPARATIVE EXAMPLE 5 5

(1) Method to manufacture a container for the measurement of cellular functions:

 The vacuum-collected blood collection tubes containing heparin and PHA-P were manufactured in the same manner as in Example 22 (1) for use as the containers for the measurement of cellular functions, except that the tubes for the collection of blood subjected to vacuum of 4 ml (12.6 x 75 mm in diameter) made of polyethylene terephthalate, different from those used in Example 22, were used instead of the tubes for collecting blood subjected to the vacuum of 4 ml (12.6 x 75 mm in diameter) made of polyethylene terephthalate as used in Example 22. 15

(2) Determination of endotoxin content in blood collection tubes (Containers):

 The endotoxin content in each blood collection tube was determined in the same manner as in Example 22 (2), except that the four vacuum-collected blood collection tubes that were obtained in the previous one were used ( 1) containing heparin and PHA-P. The results indicated the endotoxin content in the liquids extracted respectively in the four blood collection tubes tested which were 0.65 EU / ml, 0.74 EU / ml, 0.58 EU / ml and 0.62 EU / ml, respectively.

(3) Methods to determine the induction activities of TNFα, IL-1β and IL-25 6:

 The induction activities of TNFα, IL-1β and IL-6 were determined in the same manner as in (3) of Example 22, except that 10 of the vacuum-collected blood collection tubes obtained in the above were used ( 1) containing heparin and PHA-P, instead of using the tubes 30 for vacuum collection of blood prepared in (3) of Example 22 containing heparin and PHA-P. The results are shown in Tables 1 - 3.

Table 1

 Ex. 22 Ex. Comp. 5

 Induced TNFα level (pg / ml)

 Blood collection tube No. 1 452.8 684.5

 2

 420.5 790.5

 3

 468.5 948.6

 4

 440.2 664.5

 5

 495.6 895.3

 6

 462.8 980.5

 7

 442.1 728.6

 8

 432.8 894.2

 9

 489.2 925.1

 10

 438.8 696

 Mean value (pg / ml)

 454.33 820.78

 SD * (pg / ml)

 24.5 120.9

 CV * (96)

 5.4 14.7

 * SD = Standard Deviation CV = Coefficient of variation

Table 2

 Ex. 22 Ex. Comp. 5

 Induced IL-1β level (pg / ml)

 Blood collection tube No. 1 274.6 496.3

 2

 254.5 582.1

 3

 268.4 396.1

 4

 252.4 557.9

 5

 236.8 689.3

 6

 257.1 325.6

 7

 249.6 445.8

 8

 262.8 625.4

 9

 257.1 322.4

 10

 244.6 555.8

 Mean value (pg / ml)

 255.79 499.72

 SD * (pg / ml)

 11.1 124.9

 CV * (96)

 4.3 25.0

 * SD = Standard Deviation

 CV = Coefficient of variation

Table 3

 Ex. 22 Ex. Comp. 5

 Induced IL-6 level (pg / ml)

 Blood collection tube No. 1 6820 8820

 2

 6930 12520

 3

 6240 9630

 4

 7150 13050

 5

 6420 9420

 6

 6390 8420

 7

 6930 7430

 8

 6520 12490

 9

 6480 10060

 10

 6610 9420

 Mean value (pg / ml)

 6654 10126

 SD * (pg / ml)

 300 1910

 CV * (96)

 4.5 18.9

 * SD = Standard Deviation CV = Coefficient of variation

 As can be seen from Tables 1-3, the use of containers for the measurement of cellular functions, which contain 5 endotoxin in concentrations not exceeding 0.5 EU / ml per extracted liquid, provided a better reproducibility.

EXAMPLES 23 - 30 (Reference)

(1) Method for manufacturing a container for the measurement of cellular functions: 10

 Each of the 4 ml (12.6 x 75 mm diameter) blood collection tubes made of polyethylene terephthalate was washed well 10 times with 4 ml of endotoxin-free water (manufactured by Otsuka Seiyaku Co.). Sodium heparin (manufactured by Novo.Nordisk A / S Co., product name: Novo.Heparin # 1000) and PHA-L (manufactured by Sigma Chemical Co.) were dissolved in a physiological saline solution (manufactured by Otsuka Seiyaku Co .) to prepare physiological saline solutions containing 40 U / ml of sodium heparin and PHA-L at the concentration shown in Table 4, respectively. 1 ml of the respective physiological saline solutions was added to two respective blood collection tubes that had been washed previously.

 Next, a cap made of butyl rubber, which was configured to fit the tube and had been thoroughly washed with an endotoxin-free water (manufactured by Otsuka Seiyaku Co.), was placed superimposed on one opening of each tube for collecting blood so as not to tightly close the opening. Next, each blood collection tube was placed inside a vacuum vessel. When the pressure inside the vessel was gradually reduced to a pressure of 570 mm Hg, the opening of each blood collection tube was tightly closed 10 by the cap. The blood collection tubes prepared in this way were used as the vessels for the measurement of cellular functions in Examples 23-30, respectively.

(2) Determination of endotoxin content in blood collection tubes (Containers): 15

 1 ml of endotoxin-free water (manufactured by Otsuka Seiyaku Co.) was introduced into some respective (8 in total) of the containers for the measurement of cellular functions that were obtained in the previous one (1) containing various concentrations of PHA -L for further stirring at 37 ° C for 1 hour to extract the endotoxin. Next, the content of endotoxin in each extracted liquid was determined in the same manner as in Example 1. The results indicated that the endotoxin content in the extracted liquid was not greater than 0.05 EU / ml for each of the containers for the measurement of cellular functions.

(3) Methods to determine the induction activities of TNFα and IL-1β: 25

 Using an injector with a needle, heparinized blood was collected from a usually healthy volunteer. The injector needle was inserted into the cap made of butyl rubber, placed on each of the containers for measuring the cellular functions of Examples 23-30 respectively obtained in the previous one (1), to inject 1.0 ml of the blood sample collected in 30 each of the blood collection tubes. Next, each of the blood collection tubes was mounted on a stirring platform for flip mixing in a preheated thermostatic chamber at a temperature of 37 ° C for subsequent mixing by flipping for 2 hours. Once the intimate mixing was completed, each vessel was centrifuged at 1600 G at 4 ° C for 10 minutes to collect a supernatant plasma. The content (pg / ml) of TNFα and IL-1β was determined in the collected plasma in the same manner as in (3) of Example 1. The results are shown in Table 4.

 5

Table 4

 Inductor and its concentration (µg / ml) Cytokine production (pg / ml)

 PHA-L

 PHA-P TNFα IL-1β

 Ex.

 23 0.02-5

 24

 0.2 - 70 80

 25

 2 - 2178 403

 26

 10 - 2864 563

 27

 50 - 2956 567

 28

 100 - 2431 432

 29

 200 - 2250 425

 30

 500 - 153

 31

 - 0.2 25

 32

 - 2 63

 33

 - 10 164

 3. 4

 - 50 496

EXAMPLES 31 - 34 (Reference)

 The procedure practiced in Example 23 was repeated to quantitatively determine the TNFα and IL-1β produced, except that 10 PHA-P (manufactured by Sigma chemical Co.) was used at the concentration shown in Table 4, instead of use PHA-L as in Example 23. The results are shown in Table 4. The endotoxin content in the liquids, which were respectively extracted in containers for the measurement of cellular functions in Examples 31-34 in the same manner. which was performed in (2) of Example 23, were not greater than 0.05 EU / ml.

EXAMPLE 35 (Reference) AND COMPARATIVE EXAMPLE 6

 A tube for the collection of vacuum blood available on the market, that is a tube for the collection of LPS-free blood (manufactured by Sekisui chem. Ind. Co .: provided with a non-LPS specification) was used as the 20 container for the measurement of cellular functions according to the first invention (Example 35). In addition, another commercially available vacuum blood collection tube, ie a blood collection tube (not provided with a non-LPS specification) manufactured by company A, was used as a comparative blood collection tube ( Comparative Example 6).

 For each Example, blood was collected under vacuum from a usually healthy human being (same person) in five tubes for blood collection (1 ml each). Each tube for blood collection was stored at 20 ° C for 2 hours, and then centrifuged at 4 ° C (1,600 G, 10 minutes) 10 for subsequent plasma collection. The amount of TNFα in the collected plasma was determined using a product called "PREDICTA Human TNFα ELISA KIT" manufactured by Genzyme Inc. The determined values were averaged to obtain a final value for each Example. The results indicated that the concentration of TNF-α determined for Example 35 # 15 was greater than 15 pg / ml, that is, a detection limit, while for Comparative Example 6 it was 51 pg / ml.

 The blood collection tubes separated from the same lot as the LPS-free blood collection tubes for use in Example 35 and those from the same lot as the blood collection tubes manufactured by company A for use in Comparative Example 6, its endotoxin content was determined respectively as follows. 1 ml of an endotoxin-free water (manufactured by Otsuka Seiyaku Co.) was added to each of the blood collection tubes specified above for further stirring at 37 ° C for 1 hour to extract the endotoxin. Next, the endotoxin content in the liquid extracted in each tube was determined in the same manner as in Example 1. As a result, the determined endotoxin contents were 0.03 EU / ml in the collection tube for use in Example 35 and 958 EU / ml in the collection tube for use in Comparative Example 6. 30

 As is evident from the above, in determining the concentration of TNFα in blood, unless blood is collected using the blood collection vessel in which the amount of endotoxin, when extracted by collecting water free of Endotoxin a volume equal to the volume of liquid to be measured, translates into an insufficient level to induce the production of physiologically active substances from the blood cells, the blood reacts with LPS in the blood collection vessel to produce (induce) physiologically active substances such as TNFα, as well as undergo unnecessary stimulation to gradually change their properties, and as a result of the same the precise determination of the concentration of TNFα in blood per se is hindered.

EXAMPLE 36 (Reference) AND COMPARATIVE EXAMPLES 7 - 9

 A solution of LPS at a concentration of 120 ng / ml, as a stimulator, is distributed in 5 tubes for blood collection for use in Example 35, 5 tubes for blood collection for use in Comparative Example 6 , 5 tubes for the collection of vacuum blood that incorporate heparin available on the market manufactured by company B and 5 tubes for the collection of vacuum blood that incorporate heparin available on the market manufactured by company C, ie 50 µl of LPS 15 solution for each tube for blood collection, to prepare reactors to react to LPS with blood to induce cytokines. Reactors prepared using the blood collection tubes of Example 35 were assigned to Example 36, and those of Comparative Example 6 to Comparative Example 7, respectively. Reactors prepared using the blood collection tubes 20 manufactured by company B were assigned to Comparative Example 8, and those by company C to Comparative Example 9. Meanwhile, in the blood collection tubes separated from the same batch that the blood collection tubes manufactured by company B from which the reactors of Comparative Example 8 were prepared, their endotoxin content was determined separately in the same manner as in Example 35. Similarly, in the blood collection tubes separated from the same batch as the blood collection tubes manufactured by company C from which the reactors of Comparative Example 9 were prepared, the endotoxin content 30 of the same way as in Example 35. The results indicated that the blood collection tubes manufactured respectively by companies B and C, for the respective use in Comparative Examples 8 and 9, contained endotoxin in concentrations of 10 EU / ml and 389 EU / ml, respectively. 35

 Total 6 ml of blood from the heart was collected from 5 male mice, 8-10 weeks of age using an injector for subsequent injection into a blood collection vessel identical to those used in Example 35.

 The injector had previously been soaked in a 0.2 M aqueous solution of sodium hydroxide overnight to deactivate the endotoxin, washed sufficiently with endotoxin-free water, and further loaded with heparin in such an amount to allow the blood collected afterwards in the injector finally contains heparin at a concentration of 10 U / ml.

 Next, a part of the needle of a multi-needle injector 10 was nailed in a cap of the container for the collection of anterior blood and another part of the needle was nailed in a cap after another cap for the reactors for use respective in Example 36 and Comparative Examples 7-9 for distributing the collected blood in those reactors, ie 300 µl of collected blood for each reactor. fifteen

 Each reactor was then kept under stirring at 37 ° C for 4 hours, and then centrifuged (1,600 G, 10 minutes) at 4 ° C for subsequent plasma collection. The amount of TNF-α in the collected plasma was determined using a product called "FACTOR TEST MOUSE TNF-α ELISA KIT" manufactured by Genzyme Inc. The determined values were averaged 20 to obtain a final value for each Example. In addition, the coefficient of variation (%) [= (standard deviation) / (average value) x 100) was calculated for each Example. The results are given in Table 5.

Table 5 25

 TNFα production

 Mean value (pg / ml)

 CV * (%)

 Ex 36

 1036 3.7

 Ex. Comp. 7

 5610 23.7

 Ex. Comp. 8

 1078 15.1

 Ex. Comp. 9

 2961 10.5

 * CV = Variation Coefficient

 As can be seen from Table 5, the smallest variance (coefficient of variation) in the production of TNF-α (induced) was observed in Example 36 in which a known amount of LPS was added to the container for collecting LPS free blood.

 In view of the previous results, in order to establish a measurement method by which the morbidity of the patient and the others can be decided accurately and in a simplified manner, it is considered essential to store the collected blood without exposing it to unnecessary stimulation for a period of time. from collection to subject to measurements. To this end, it is necessary, when collecting blood, to use a blood collection vessel that has an interior with reduced pressure and contains LPS at an insufficient level to allow physiologically active substances (released or induced) to be produced, as can be clearly seen.

 It is also evident from the previous results, that to accurately measure the functions of blood cells, it is necessary to use a blood collection vessel that is not contaminated with endotoxin at a concentration sufficient to adversely affect the 15 measured values. It is also necessary to distribute the collected blood in blood collection containers each containing a predetermined amount of stimulator to react in this way to the blood collected with the stimulator for the production (release or induction) of the physiologically active substances that subsequently, they are determined quantitatively.

EXAMPLE 37 (Reference)

 The instruments and containers for use in this Example were exposed to dry heating at 250 ° C for 2 hours or more, if they were made of glass, or alternatively, they were soaked in a 0.2 25 M aqueous solution of sodium hydroxide overnight to deactivate the endotoxin and washed sufficiently with endotoxin-free water, if they were made of plastic. In addition, this operation was carried out in a clean workbench.

 10,000 units of sodium heparin (manufactured by Wako Junyaku Co.) were dissolved in 10 ml of physiological saline for injection (manufactured 30 by Otsuka Seiyaku Co.). The resulting sodium heparin solution was subjected to ultrafiltration with centrifugation at 500 G at 4 ° C for 1 hour, using a CENTRIPREP 100 ultrafilter (0.1 million fractional molecular weight, manufactured by Amicon Co.). The ultrafilter for use had previously been soaked in a 0.2 M aqueous solution of sodium hydroxide overnight to deactivate the endotoxin and washed sufficiently with the endotoxin-free water.

 In the ultrafiltered sodium heparin solution, the endotoxin content was determined using ENDOSPECIE ES-6 (manufactured by Seikagaku Ind. Co.). The results indicated that the content of endotoxin in the sodium heparin solution was 0.01 EU / heparin unit.

 Next, 0.1 ml of the ultrafiltered sodium heparin solution (1,000 U / ml) was added to a 10 ml blood collection injector (manufactured by Terumo Co.), supplemented with 10 ml of blood collected from a 10 usually healthy human volunteer (final blood heparin concentration of approximately 10 U / ml). A fraction of blood immediately after collection, as well as another fraction of blood that was allowed to stand in a thermostatic chamber at 37 ° C for 2 hours and 4 hours, were centrifuged respectively at 1,600 G at 4 ° C for 10 minutes to collect a supernatant plasma 15 for each one.

 In the collected plasma, the cytokine content, ie the contents of TNFα, IL-1β and IL-6, was determined using enzyme immunoassay kits containing respective monoclonal antibodies against those cytokines. The content of each cytokine was determined using n = 3, and the average value obtained from them is shown in Table 6.

COMPARATIVE EXAMPLE 10

 Blood collection was performed in the same manner as in Example 37, except that the sodium heparin solution used was not subjected to ultrafiltration treatment. In the collected plasma, the content of cytokines, i.e. the content of TNFα, IL-1β and IL-6, was determined using enzyme immunoassay kits containing respective monoclonal antibodies against those cytokines. The content of each cytokine was determined using n = 3, and the average value obtained from them is shown in Table 6. 30

 In addition, in the sodium heparin solution used in this Comparative Example 10 the endotoxin content was determined in the same manner as in Example 37. The indicated results were 1.2 EU / heparin unit.

 35

Table 6

 * Immediately (pg / ml) * 2 hours (pg / ml) * 4 hours (pg / ml)

 Ex 37

 TNFα 35 35 35

 IL-1β

 15 15 15

 IL-6

 35 35 35

 Ex. Comp. 10

 TNFα 35 2850 ± 298 6580 ± 596

 IL-1β

 15 548 ± 42 3490 ± 412

 IL-6

 35 1225 ± 179 4865 ± 329

 * Period of time after blood collection

 As is evident from Table 6, in the system using the ultrafiltered sodium heparin solution in which the endotoxin content was reduced to 0.01 EU / heparin unit (endotoxin concentration in blood of approximately 0, 1 EU / ml), TNFα, IL-1β and IL-6 were not produced in an appreciable amount until 4 hours had elapsed since blood collection. On the other hand, in the system using the untreated sodium heparin solution in which the endotoxin content was 1.2 EU / heparin unit (blood endotoxin concentration of approximately 12 Eu / ml), due to the presence of an appreciable amount of endotoxin, increasing amounts of TNFα, IL-1β and IL-6 occurred in blood over time.

EXAMPLES 38 - 40 (Reference)

 The instruments and containers for use in this Example were exposed to dry heating at 250 ° C for 2 hours or more, if they were made of glass, or alternatively, soaked in a 0.2 M aqueous solution of sodium hydroxide overnight. to deactivate the endotoxin and washed sufficiently with endotoxin-free water, if they were made of plastic. In addition, the operation was carried out in a clean workbench. twenty

 A 0.01 ml solution containing 10 units of sodium heparin prepared in Example 37 containing endotoxin at a concentration of 0.01 EU / unit of heparin was added to each tube for the collection of free 5 ml blood. of endotoxin (12.6 x 75 mm in diameter, manufactured by Sekisui Chem. Ind. Co.) made of polyethylene terephthalate. 25

 Then, standard endotoxin obtained from the E. coli strain UKT-B of the Japanese Pharmacopoeia was dissolved in 1.6 ml of a physiological saline solution for injection for subsequent stage dilution. Once each dilution stage is completed, the resulting physiological saline solution with dissolved endotoxin is added to the tubes for blood collection with incorporated sodium heparin, so that the tubes for blood collection containing endotoxin in concentrations respectively are prepared of 0.1 EU / ml (Example 38), 0.2 EU / ml (Example 39) and 0.4 EU / ml (Example 40) by collected blood.

 Next, a cap made of butyl rubber, which was configured to fit in the blood collection tube, was placed superimposed on an opening of each blood collection tube so as not to hermetically close the opening. Next, each blood collection tube was placed inside a container whose pressure could be reduced. When the pressure inside the vessel was reduced to a pressure sufficient for the suction of 1 ml of blood, the opening of each tube for the collection of blood was sealed tightly by means of the cap.

 Next, blood was collected by vacuuming a usually healthy volunteer in the blood collection tubes, that is 1 ml each. Each of the blood collection tubes was mounted on a stirring platform for mixing by flipping in a preheated thermostatic chamber 20 at a temperature of 37 ° C for subsequent mixing by flipping for 2 hours. Once the intimate mixing was completed, each tube was centrifuged at 1,600 G at 4 ° C for 10 minutes to collect a supernatant plasma.

 The cytokine content was determined in the collected plasma, that is, the respective content of TNFα, IL-1β and IL-6 in the same manner as in Example 37. The determination of the content of each cytokine was performed using n = 3 , and the average value obtained from them is shown in Table 7.

COMPARATIVE EXAMPLE 11, 12 30

 The procedure of Example 38 was repeated to collect blood, except that the physiological saline solution with dissolved dissolved endotoxin was added to the blood collection tubes with incorporated sodium heparin, so that the blood collection tubes containing respectively endotoxin in concentrations of 0.5 EU / ml (Comparative Example 11) and 1.0 EU / ml (Comparative Example 39) per tube for blood collection.

 In the collected plasma the cytokine content was determined, that is, the respective content of TNFα, IL-1β and IL-6 in the same manner as in Example 37. The determination of the content of each cytokine was performed using n = 3 , and the average value obtained from them is shown in Table 7.

Table 7

 * Endotoxin (EU / ml)

 TNFα (pg / ml) IL-1β (pg / ml) IL-6 (pg / ml)

 Ex.

 38 0.1 35 15 35

 39

 0.2 35 15 35

 40

 0.4 35 15 35

 Ex. Comp.

 11 0.5 485 ± 29 154 ± 12 189 ± 21

 12

 1.0 3560 ± 398 1548 ± 162 2225 ± 279

 * Concentration in blood

 10

 As is evident from Table 7, if the blood endotoxin content exceeds 0.5 EU / ml, it induces productions of TNFα, IL-1β and IL-6.

EXAMPLE 41

<Method for manufacturing a kit for measuring cell functions>

 Sodium heparin (manufactured by Novo.Nordisk A / S Co., product name: NOVO.HEPARIN # 1000) was diluted with an endotoxin-free water (manufactured by Otsuka Seiyaku Co.) to obtain a sodium heparin solution containing heparin at a concentration of 200 U / ml. 0.05 ml of the sodium heparin solution was added to the blood collection tube, which had been prepared in Example 1 containing endotoxin at a concentration not exceeding 0.05 EU, so that the tube for Blood collection contained 10 Units of heparin. The resulting solution was dried under vacuum. Next, a cap made of butyl rubber, which was configured to fit in the blood collection tube, was placed superimposed on an opening of the blood collection tube so as not to tightly close the opening. The blood collection tube was then placed inside a container whose pressure can be reduced. When the pressure inside the vessel was reduced to a pressure sufficient for the suction of 1 ml of blood, that is to say a pressure of 570 mm Hg, the opening of the blood collection tube was sealed tightly by means of the cap. In this way, the first container for measuring cellular functions was manufactured. 5

 Sodium heparin (manufactured by Novo.Nordisk A / S Co., product name: NOVO.HEPARIN # 1000) was diluted with an endotoxin-free water (manufactured by Otsuka Seiyaku Co.) to obtain a solution of sodium heparin containing heparin at a concentration of 200 U / ml. Meanwhile, endotoxin obtained from strain 055: B5 of E. Coli. (manufactured by LBL Co.) was diluted with an endotoxin-free water to obtain a solution with dissolved endotoxin containing endotoxin at a concentration of 2,000 EU / ml. 0.05 ml of the sodium heparin solution was added to the blood collection tube, which had been prepared in Example 1 containing endotoxin at a concentration not exceeding 0.05 EU, so that tube 15 for Blood collection contained 10 Units of heparin. 0.05 ml of the solution with dissolved endotoxin was subsequently added to the blood collection tube. The resulting solution was dried under vacuum. Next, a plug made of butyl rubber was placed superimposed on an opening in the blood collection tube so as not to hermetically close the opening. The blood collection tube 20 was then placed inside a container whose pressure can be reduced. When the pressure inside the vessel was reduced to a pressure sufficient for the suction of 1 ml of blood, that is to say a pressure of 570 mm Hg, the opening of the blood collection tube was sealed tightly by means of the cap. In this way the 25 second vessel was manufactured for the measurement of cellular functions.

 The first and second reagents for the determination of TNFα were manufactured according to the following procedures.

 Human anti-TNFα monoclonal antibody (manufactured by Genzyme Inc.) was diluted with a 0.1 M carbonate buffer (pH 9.5) to obtain a diluted solution at a concentration of 1 µg / ml (immobilized antibody solution for first TNFα determination reagent, that is for determination with high sensitivity) and a diluted solution at a concentration of 100 ng / ml (immobilized antibody solution for the second TNFα determination reagent, that is for determination with low sensitivity), respectively. 100 100 µl of each diluted solution was added to each well of a 96-well microplate (Nunc MaxiSorp microtiter plates) for subsequent incubation at 2-8 ° C for one day and one night. Each well was then washed by suction 3 times with a phosphate buffer (pH 7.3) containing 0.05% by weight Tween 20. Next, 250 µl of a phosphate buffer 5 (pH 7.3) containing 4% by weight bovine serum albumin (manufactured by Sigma Chemical Co.) was added to each well for subsequent incubation at 37 ° C for 2 hours. The incubated solutions were then removed from each well of the microplate that was subsequently dried at room temperature. 10

 In addition, commercially available reagents were used for human TNFα (manufactured by Genzyme Inc.), viotin-labeled human anti-TNFα polyclonal antibody (manufactured by Genzyme Inc.), hydrogen peroxide, and a substrate solution containing tetramethylbenzidine ( manufactured by KPL Co.). In addition, a phosphate buffer (pH 7.3) containing 0.05% by weight of 15 Tween 20 was used as a wash solution. A 2M solution of sulfuric acid was prepared for use as a stop solution.

<Measurement of TNFα production capacities>

 An injector with a needle was used to collect heparinized blood from a usually healthy volunteer. The needle was successfully nailed in caps 20 made of butyl rubber for the respective first and second containers for the measurement of cellular functions to inject 1 ml of sample blood into each container. Next, each of the vessels that house blood was mounted on a stirring platform for flip mixing placed in a preheated thermostatic chamber at a temperature of 37 ° C for subsequent flipping mixing for 4 hours. Once the intimate mixing was completed, each tube was centrifuged at 1,600 G at 4 ° C for 10 minutes to collect a supernatant plasma. Plasma collected from the first vessel for the measurement of cellular functions was distributed in 4 wells for the determination of TNFα content in the same manner as in (3) of Example 1, using the high sensitivity reagent specified above. On the other hand, the plasma collected from the second vessel for the measurement of cellular functions was distributed in 4 wells for the determination of the TNFα content in the same manner as in (3) of Example 1, using the low sensitivity reagent specified above. . 35

COMPARATIVE EXAMPLE 13

 The production of TNFα was induced in the same manner as in Example 41 in each of the first and second vessels for the measurement of cellular functions, and the plasma collected from each of them was distributed in 4 wells for the determination of the content of plasma TNFα using a commercially available kit (PREDICTA Human TNF-α KIT). When the measured values exceeded its detection limit, the collected plasma was diluted with a phosphate buffer (pH 7.3) containing 1% by weight of bovine serum albumin at a dilution rate of approximately 5 for another measurement. The TNFα contents were measured by multiplying the measured values 10 by the dilution ratio.

 The results of Example 41 and Comparative Example 13 are given in the following Table 8.

Table 8 15

 Ex. 41 [Concentration of TNFα in plasma (pg / ml: Mean value ± SD, CV (%))]

 No. 1

 No. 2 No. 3 No. 4

 2nd Container for the measurement of cellular functions

 3870 ± 280, 7.2 2570 ± 180, 7.0 4870 ± 380, 7.8 4520 ± 340, 7.5

 1st container for measuring cell functions

 <* LD <* LD <* LD <* LD

 Ex. Comp. 13 [Concentration of TNFα in plasma (pg / ml: Mean value ± SD, CV (%))]

 No. 1 No. 2 No. 3 No. 4

 2nd Container for the measurement of cellular functions

 1520 ± 400, 15.8 2070 ± 310, 14.9 4510 ± 620, 13.7 4020 ± 780, 19.4

 1st container for measuring cell functions

 <* LD <* LD <* LD <* LD

 * LD = Detection Limit

 In Table 8, CV indicates a coefficient of variation.

 As is clear from the results in Table 8, the commercial kit cannot be used in the determination of the amount of TNFα induced in the second vessel for the measurement of cellular functions 5 before the dilution operation is performed. The Example gave the results with greater reproducibility, that is the coefficients of variation of approximately 7.5%. On the other hand, the Comparative Example, in which the measurement of the contents of TNFα was carried out by means of the commercial kit after plasma dilution, gave the results with less reproducibility, that is, the coefficients of variation varied between approximately 15 and approximately 20%.

EFFECTS OF THE INVENTION

 According to the present application, in the first container for the measurement of cellular functions, for use in the determination of 15 physiologically active substances produced from blood cells, the container is characterized in that the amount of material capable of inducing the The production of physiologically active substances, when extracted by collecting water of a volume equal to a volume of liquid to be measured, is limited to an insufficient level to induce the production of physiologically active substances from blood cells. Therefore, the collected blood is hardly subjected to unnecessary stimulation for a period from collection to measurement so that long-term preservation of it is allowed. This allows the precise measurement of the physiologically active substances present in the collected blood and allows the use of the container in the examination in a precise way of morbidities of patients who have various diseases.

 In addition, the first container for the measurement of cellular functions according to the invention can be suitably used to obtain 5 control values, when used in combination with the second container for the measurement of cellular functions.

 In the first container for the measurement of cellular functions according to the present application, (the material capable of inducing the production of the physiologically active substances described above is endotoxin), 10 the endotoxin content in the container for the measurement of Cellular functions before use is preferably controlled so that it does not exceed 0.5 EU / ml as a concentration in an extracted liquid when it is extracted by collecting water of a volume equal to a volume of liquid to be measured. This allows the precise measurement of the physiologically active substances present in the collected blood without being altered by the endotoxin content in the container before the blood collection.

 In the second container for the measurement of cellular functions according to the invention, although the material capable of inducing the production of physiologically active substances in blood is housed in such a way that it can come into contact with the blood, the content of the Material capable of inducing the production of physiologically active substances, originally present in the container before being housed, is limited to an insufficient level to influence the measured values of physiologically active substances. Accordingly, the production of physiologically active substances can be determined very precisely when blood is introduced and comes into contact with the material capable of inducing the production of physiologically active substances to thereby produce physiologically active substances. .

 In addition, in the second container for the measurement of cellular functions according to the invention, (the material that induces the production of the physiologically active substances described above is endotoxin), the concentration of endotoxin in a resulting complete liquid when placed in contact with blood is limited, being in the range of 0.6 - 100,000 EU / ml. Accordingly, the production of physiologically active substances can be determined very precisely when blood is introduced and comes into contact with the material capable of inducing the production of physiologically active substances to thereby produce physiologically active substances. .

 In the first or second container for the measurement of cellular functions, the additional housing of anticoagulants serves to prevent blood clotting in the container for the measurement of cellular functions.

 In addition, when the amount of material capable of inducing the production of physiologically active substances contained in the anticoagulant described above is controlled at an insufficient level to induce the production of physiologically active substances from blood cells when mixed with blood, The physiologically active substances produced can be determined very precisely without being influenced by the materials that induce physiologically active substances originally contained in the anticoagulant.

 In addition, in the first or second container for the measurement of cellular functions, the reduction of the pressure inside the container facilitates the introduction of blood into the container for the measurement of cellular functions. Therefore, operations that include manually transferring blood to various reactors such as pipetting subsequently to the collection of blood from an examined human being, cell separation, cell culture and the like are not required. This eliminates the risk that a person performing the test will contract various infectious diseases, such as hepatitis and AIDS. In addition, the content of endotoxin 25 in the container prior to use is limited to thereby eliminate a possibility that various bacteria or dusts are accidentally incorporated into a blood sample. The potential problem of causing unnecessary activation or drop in cell activation due to the presence of contaminants or performing various operations is thus avoided. 30 In addition, since whole blood is used, specialized techniques that include cell separation and culture, microscopic measurement and so on are not required. This shortens the period of time for measurement and eliminates the need to introduce RI facilities and expensive equipment such as a flow cytometer. As a result, a measurement of cell functions can be performed that is simpler, less expensive and more accurate than conventional operation.

 In the kit for the measurement of cellular functions according to the present invention which includes the first and second containers for the measurement of cellular functions according to and the reagent capable of quantitatively determining the physiologically induced substances induced, the use of the reagent Quantifiable defined above allows easy quantification of induced physiologically active substances.

 In addition, the present application includes the container for the measurement of cellular functions, the second container for the measurement of cellular functions, and the reagent for quantitatively determining physiologically active substances. Therefore, when blood is introduced into both the first and second containers for the measurement of cellular functions, for quantification by the reagent specified above, the control value can be obtained from the first container 15 for the measurement of the cellular functions while the measured values corresponding to the amount of physiologically active substances induced in blood by the inducing material of physiologically active substances. As a result, highly accurate quantification of physiologically active substances in blood is allowed. twenty

 In such a case, the first and second enzymatic immunoassay reagents that have different sensitivities from each other can be used for the first and second containers for the measurement of cellular functions, respectively, in accordance with the intended purposes thereof. This allows precise quantification of the physiologically active substances induced.

 In addition, in the kit for measuring cellular functions according to the invention, (the inducing material of physiologically active substances is endotoxin), the content of endotoxin in the second container is controlled so that the concentration of endotoxin in a liquid Complete 30 resulting when contacted with blood is in the range of 0.6 - 100,000 EU / ml. As a result, the amount of physiologically active substances produced in blood when the endotoxin came into contact with the blood can be determined very precisely.

 In the method for the measurement of cellular functions, blood is introduced into the second vessel for the measurement of cellular functions, and is brought into contact with the inducing material of physiologically active substances to induce physiologically active substances. Since the content of endotoxin in the second container for the measurement of cellular functions is limited to the range specified above, the amount of physiologically active substances produced in blood can be determined very precisely.

 In this case, the production of the physiologically active substances can be reliably induced by inducing the production of physiologically active substances at a temperature in the range of 26-45 ° C, or as an alternative, inducing the production of physiologically active substances in the period of time of 1 - 6 hours.

 In the method for measuring cellular functions, the amount of physiologically active substances produced in blood can be determined reliably by quantifying the physiologically active substances using the reagent capable of quantitatively determining them.

Claims (3)

1. A kit for the measurement of cellular functions comprising: (a) a first container for the measurement of cellular functions for use in determining a cytokine produced by blood cells, in which an amount of endotoxin, when extracted by collecting water of a volume equal to a volume of liquid to be measured, before use does not exceed 0.5 EU / ml, and an anticoagulant is housed. (b) a second vessel for the measurement of cellular functions in which endotoxin and anticoagulant are housed so that they can be contacted with blood, in which the concentration of endotoxin in a resulting complete liquid when placed in contact with blood in the second vessel for the measurement of cellular functions, translates into the range of 0.6 - 100,000 EU / ml; Y (c) a reagent for quantifying said cytokine, wherein said reagent 15 includes: a first enzyme immunoassay reagent having a high measurement sensitivity of 10-1000 pg / ml used to quantitatively determine the cytokine in the blood collected in the first container for the measurement of cellular functions; twenty a second enzyme immunoassay reagent that has a low measurement sensitivity of 500-10,000 pg / ml used to quantitatively determine the cytokine produced by reacting the endotoxin with the blood collected in the second vessel for the measurement of cellular functions, having the second enzyme immunoassay reagent a different measurement sensitivity, for the cytokine, to that of the first enzyme immunoassay reagent. 2. The kit for measuring cellular functions according to claim 1, said first and second containers comprising a container body 30 having an opening and a cap fixed to seal the opening, which is not removed during the collection of the blood. 3. The kit for measuring cell functions according to any one of claims 1 to 2, wherein in each interior of said first and second containers for measuring cell functions the pressure has been reduced.